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Candida albicans remains the main causal agent of candidiasis, the most common fungal infection with disturbingly high mortality rates worldwide. The limited diversity and efficacy of clinical antifungal drugs, exacerbated by emerging drug resistance, have resulted in the failure of current antifungal therapies. This imposes an urgent demand for the development of innovative strategies for effective eradication of candidal infections. While the existing clinical drugs display fungicidal or fungistatic activity, the strategy specifically targeting C. albicans filamentation, as the most important virulence trait, represents an attractive approach for overcoming the drawbacks related to clinical antifungals. The results acquired in this study revealed the significant potential of 5-aminotetrazoles as a new class of effective and safe anti-virulence agents. Moreover, these novel agents were active when applied both alone and in combination with clinically approved polyenes. Complete prevention of C. albicans morphogenetic yeast-to-hyphae transition was achieved at doses as low as 1.3 μM under conditions mimicking various filamentation-responsive stimuli in the human body, while no cardio- or hepatotoxicity was observed at doses as high as 200 μM. The treatment of C. albicans-infected zebrafish embryos with nystatin alone had low efficacy, while the combination of nystatin and selected 5-aminotetrazoles prevented fungal filamentation, successfully eliminating the infection and rescuing the infected embryos from lethal disseminated candidiasis. In addition, the most potent anti-virulence 5-aminotetrazole prevented C. albicans in developing the resistance to nystatin when applied in combination, keeping the fungus sensitive to the antifungal drug.


Supplementary Material Contents: Page
Table S1 2 Figure S1 3 Figure S2 4 1 H and 13 C NMR spectra of representative products 5

HPLC purity chromatograms 14
Table S1 Supplementary Table S1.Lethal and teratogenic effects observed in zebrafish (Danio rerio) embryos at different hours post fertilization (hpf).
a No clear organs structure is recognized.b Malformation of eyes was recorded for the retardation in eye development and abnormality in shape and size.c Presence of none, one or more than two otoliths per sacculus, as well as reduction and enlargement of otoliths and/or sacculi (otic vesicles).d Tail malformation was recorded when the tail was bent, twisted or shorter than to control embryos as assessed by optical comparation.
e Growth retardation was recorded by comparing with the control embryos in a body length (after hatching, at and onwards 72 hpf) using by optical comparison using an inverted microscope (CKX41; Olympus, Tokyo, Japan).

Teratogenic effect
Malformation of head Growth retardation

Hepatotoxicity
Yolk absorption
Eluent was made from the following solvents: 0.2% formic acid in water (A) and methanol (B).The analysis were performed at the UV max of the compounds (at 254 or 330 nm) to maximize selectivity.
Eluent was made from the following solvents: 0.2% formic acid in water (A) and acetonitrile (B).The analysis were performed at the UV max of the compounds (at 254 or 330 nm) to maximize selectivity.
Eluent was made from the following solvents: 0.2% formic acid in water (A) and methanol (B).The analysis were performed at the UV max of the compounds (at 254 or 330 nm) to maximize selectivity.
Eluent was made from the following solvents: 0.2% formic acid in water (A) and acetonitrile (B).The analysis were performed at the UV max of the compounds (at 254 or 330 nm) to maximize selectivity.