A Screening Assay Based on Host-Pathogen Interaction Models Identifies a Set of Novel Antifungal Benzimidazole Derivatives

Abstract

Fungal infections are a serious health problem in clinics, especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses, only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapy, significant side effects, and high costs for several antifungals, there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities, we screened a compound library including more than 35,000 individual compounds derived from organic synthesis as well as combinatorial compound collections representing mixtures of compounds for antimycotic activity. In total, more than 100,000 compounds were screened using a new type of activity-selectivity assay, analyzing both the antifungal activity and the compatibility with human cells at the same time. One promising hit, an (S)-2-aminoalkyl benzimidazole derivative, was developed among a series of lead compounds showing potent antifungal activity. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole showed the highest antifungal activity and the best compatibility with human cells in several cell culture models and against a number of clinical isolates of several species of pathogenic Candida yeasts. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol pathway, in contrast to other benzimidazole derivatives, which target microtubules.

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols

“…Raw data were created using Molecular Devices GenePix Pro 7.0 imaging software (Molecular Devices, Sunnyvale, CA). The data were statistically analyzed using R software (version 2.10.1), the Limma package (version 3.2.1), and the StatMod package (version 1.4.2) (21, 22), as described previously (11,(22)(23)(24)(25). Background correction was performed using the "normexp" method, with an offset of 50 (26).…”

“…For data analysis, a "gal file" was produced using TAS Application Suite software (version 2.7.1.18; Genomic Solutions, Huntingdon, United Kingdom) (11). To avoid nonspecific binding, wholegenome C. albicans SC5314 DNA microarrays were blocked as described previously (11).…”

“…RNA isolation, preparation for microarray analysis, and hybridization were performed exactly as described previously (11).…”

“…Wholegenome C. albicans SC5314 DNA microarrays were printed as described previously (11). For data analysis, a "gal file" was produced using TAS Application Suite software (version 2.7.1.18; Genomic Solutions, Huntingdon, United Kingdom) (11).…”

“…Due to the eukaryotic nature of fungal pathogens, well-tolerated antifungals are identified much less frequently than antibiotics targeting bacteria. Using an assay that mimics the smallest unit of a natural infection through incubation of the pathogen in the presence of host cells, we previously identified a benzimidazole, EMC120B12 [(S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole] (10,11), with large antifungal effects even against resistant yeasts such as Candida krusei (12). Our initial studies using transcriptional profiling indicated that the ergosterol pathway might be the target of the new compound.…”