Identification of an Aminothiazole with Antifungal Activity against Intracellular Histoplasma capsulatum

Abstract

As eukaryotes, fungi possess relatively few molecules sufficiently unique from mammalian cell components to be used as drug targets. Consequently, most current antifungals have significant host cell toxicity. Primary fungal pathogens (e.g., Histoplasma) are of particular concern, as few antifungals are effective in treating them. To identify additional antifungal candidates for the treatment of histoplasmosis, we developed a high-throughput platform for monitoring Histoplasma growth and employed it in a phenotypic screen of 3,600 commercially available compounds. Seven hit compounds that inhibited Histoplasma yeast growth were identified. Compound 41F5 has fungistatic activity against Histoplasma yeast at micromolar concentrations, with a 50% inhibitory concentration (IC 50 ) of 0.87 M, and has the greatest selectivity for yeast (at least 62-fold) relative to host cells. Structurally, 41F5 consists of an aminothiazole core with an alicyclic substituent at the 2-position and an aromatic substituent at the 5-position. 41F5 inhibits Histoplasma growth in liquid culture and similarly inhibits yeast cells within macrophages, the actual host environment of this fungal pathogen during infection. Importantly, 41F5 protects infected host cells from Histoplasmainduced macrophage death, making this aminothiazole hit compound an excellent candidate for development as an antifungal for Histoplasma infections.

Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts

“…It is hoped that these standardized methods will provide for more reliable profiling of clinical isolates of Histoplasma as well as accelerate high-throughput screening of compounds for new antifungals active against the pathogenic phase of Histoplasma (22).…”

“…This study was motivated by the inadequacy of CLSI-defined yeast culture procedures for the growth of Histoplasma yeasts, the clinical form of Histoplasma. In some previous studies, the CLSI M27-A2 and -A3 standards have been adapted for Histoplasma yeast (22)(23)(24)(25), but no consensus methods or optimization of assay parameters have been detailed. Here, we optimize the inoculum size and assay duration to maximize the dynamic range of a microdilution assay for Histoplasma.…”

Antifungal therapeutics for dimorphic fungal pathogens

“…The top hit aminothiazole (41F5) was effective against yeasts in culture (MIC 2-4 mM) and effectively inhibited proliferation of yeasts resident within macrophages. 98 Importantly, the aminothiazole compound had low toxicity to cultured mammalian cells, including macrophages. Unfortunately, the aminothiazole was not active against Blastomyces yeasts despite the very close phylogenetic relationship between Histoplasma and Blastomyces.…”

“…To enable rapid quantitative screening of yeast growth, yeasts were engineered to express a red-fluorescent protein, which could be used as a surrogate indicator of the number of yeast cells. 98 Furthermore, fluorescence-based growth analysis facilitated efficient monitoring of Histoplasma within macrophages, and thus the determination of the effectiveness of compounds on inhibition of Histoplasma within its host cell environment. The top hit aminothiazole (41F5) was effective against yeasts in culture (MIC 2-4 mM) and effectively inhibited proliferation of yeasts resident within macrophages.…”

Eng1 and Exg8 Are the Major α-Glucanases Secreted by the Fungal Pathogen Histoplasma capsulatum

“…Cell Wall Sensitivity Assays-Yeast cells were grown in 96-well microtiter plates (38) with graded concentrations of antifungal compounds (fluconazole (Glenmark Generics); caspofungin (Merck); and nikkomycin Z (gift from John Galgiani)) and cell wall-destabilizing compounds (Calcofluor White (Sigma); Congo red (MP Biomedicals); SDS; and Uvitex) (39). Growth after 4 days was measured by optical density at 595 nm and compared with the turbidity of wells lacking inhibitors.…”