Identification of novel cell wall destabilizing antifungal compounds using a conditional Aspergillus nidulans protein kinase C mutant

Mircus, Gabriel; Hagag, Shelly; Levdansky, Emma; Sharon, Haim; Shadkchan, Yona; Shalit, Itamar; Osherov, Nir

doi:10.1093/jac/dkp270

Cited by 12 publications

(12 citation statements)

References 30 publications

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“…Alternatively, one can introduce the human HSET/KIFC1 gene into A. nidulans , and if it can functionally substitute for klpA , a bimC 4 mutant containing this human gene would be ideally suited for directly identifying HSET/KIFC1 inhibitors. A. nidulans is a well established genetic system for studying mitosis and microtubule motors in general (Morris, 1975; Enos and Morris, 1990; O’Connell et al, 1993; Oakley, 2004; Osmani and Mirabito, 2004; Xiang and Fischer, 2004; Peñalva et al, 2012; Pantazopoulou et al, 2014; Steinberg, 2014; Egan et al, 2015; Xiang et al, 2015) and A. nidulans -based drug screening has been performed previously (Kiso et al, 2004; Mircus et al, 2009; Zhai et al, 2010), further supporting the feasibility of our proposed kinesin-14 inhibitor screen.…”

Section: Discussionsupporting

confidence: 74%

The Aspergillus nidulans bimC4 mutation provides an excellent tool for identification of kinesin-14 inhibitors

Wang

Jin

et al. 2015

Fungal Genetics and Biology

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Centrosome amplification is a hallmark of many types of cancer cells, and clustering of multiple centrosomes is critical for cancer cell survival and proliferation. Human kinesin-14 HSET/KFIC1 is essential for centrosome clustering, and its inhibition leads to the specific killing of cancer cells with extra centrosomes. Since kinesin-14 motor domains are conserved evolutionarily, we conceived a strategy of obtaining kinesin-14 inhibitors using Aspergillus nidulans, based on the previous result that loss of the kinesin-14 KlpA rescues the non-viability of the bimC4 kinesin-5 mutant at 42°C. However, it was unclear whether alteration of BimC or any other non-KlpA protein would be a major factor reversing the lethality of the bimC4 mutant. Here we performed a genome-wide screen for bimC4 suppressors and obtained fifteen suppressor strains. None of the suppressor mutations maps to bimC. The vast majority of them contain mutations in the klpA gene, most of which are missense mutations affecting the C-terminal motor domain. Our study confirms that the bimC4 mutant is suitable for a cell-based screen for chemical inhibitors of kinesin-14. Since the selection is based on enhanced growth rather than diminished growth, cytotoxic compounds can be excluded.

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Section: Discussionsupporting

confidence: 74%

The Aspergillus nidulans bimC4 mutation provides an excellent tool for identification of kinesin-14 inhibitors

Wang

Jin

et al. 2015

Fungal Genetics and Biology

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“…Sixteen hits were identified and were further analyzed. We used an inducible alcA-PKC mutant of A. nidulans hypersensitive to cell wall damage in order to identify which of the 16 compounds affects cell wall integrity, as described previously (8,9). Eight compounds were identified, including five CANBEF derivatives and three other compounds that were later dropped due to toxicity for cultured mammalian cells (see Table S1 in the supplemental material).…”

Section: Discussionmentioning

confidence: 99%

“…To determine whether the selected compounds inhibit fungal growth by damaging the integrity of the cell wall, A. nidulans strain R153 and the isogenic conditional alcA-PKC mutant (8) were grown in 96-well plates at a concentration of 10 4 conidia/ml in MM or MMG (MMG contains 0.2% [wt/vol] glycerol instead of glucose). The wells were supplemented with 2-fold dilutions of each selected compound.…”

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of a Novel Family of Selective Antifungal Compounds (CANBEFs) That Interfere with Fungal Protein Synthesis

Mircus

Albert

Ben-Yaakov

et al. 2015

Antimicrob Agents Chemother

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cInvasive mycotic infections have become more common during recent decades, posing an increasing threat to public health. However, despite the growing needs, treatments for invasive fungal infections remain unsatisfactory and are limited to a small number of antifungals. The aim of this study was to identify novel fungal cell wall inhibitors from a library of small chemical compounds using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans sensitive to cell wall-active agents. Eight "hit" compounds affecting cell wall integrity were identified from a screen of 35,000 small chemical compounds. Five shared a common basic molecular structure of 4-chloro-6-arylamino-7-nitro-benzofurazane (CANBEF). The most potent compound, CANBEF-24, was characterized further and was shown to inhibit the growth of pathogenic Aspergillus, Candida, Fusarium, and Rhizopus isolates at micromolar concentrations but not to affect the growth of mammalian cell lines. CANBEF-24 demonstrated strong synergy in combination with caspofungin, an antifungal that inhibits cell wall biosynthesis. Genetic and biochemical analyses with Aspergillus nidulans and Saccharomyces cerevisiae indicated that CANBEFs selectively inhibit fungal rRNA maturation and protein synthesis, suggesting that their effect on the cell wall is indirect. CANBEFs were nontoxic in insect (Galleria mellonella, Drosophila melanogaster) and mouse models of fungal infection. Preliminary evidence showing no therapeutic benefit in these models suggests that further cycles of optimization are needed for the development of this novel class of compounds for systemic use.T he number of life-threatening invasive fungal infections has risen dramatically over the past 30 years (1, 2). The vast majority of these infections are caused by species of the genera Candida, Aspergillus, Cryptococcus, and Coccidioides (3). Invasive aspergillosis has now overtaken candidiasis as the most frequent invasive fungal infection found after death in Europe and the United States (4, 5). Today, as many as 4% of all patients dying in modern tertiary care hospitals have invasive aspergillosis caused by fungal pathogen species that belong to the genus Aspergillus, while as many as 2% of these patients suffer from invasive candidiasis caused by Candida species (4, 6). However, despite the growing needs, treatments for invasive fungal infections remain unsatisfactory.There are three main classes of antifungal drugs in common clinical use for the treatment of systemic mycoses: the polyene amphotericin B, which binds fungal membrane ergosterol, leading to cell lysis; azoles, which inhibit ergosterol biosynthesis (fluconazole, itraconazole, voriconazole [VRC], and posaconazole); and the newly introduced echinocandins, such as caspofungin (CAS), which inhibit fungal glucan biosynthesis. Most of these current systemic antifungal treatments interact unfavorably with other medications, have resistance problems, a narrow spectrum of activity, and limited formulations, and are fungistatic rather...

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“…Although A. terreus is beneficial for industrial production of lavastatin, gliotoxin and bioethanol [2], the pathogen causes severe damages in agriculture and human health [3]. Disturbingly, there is prediction that 4% of all patients who die in hospitals die of invasive aspergillosis [4]. A. terreus causes severe loss to important crops worldwide, and destroying over 125 million tons of rice ( Oryza sativa L.), wheat ( Triticum aestivum ), potato ( Solanum tuberosum L.), maize ( Zea mays ) and soyabean ( Glycine max L . )…”

Section: Introductionmentioning

confidence: 99%

Invasion of Solanum tuberosum L. by Aspergillus terreus: a microscopic and proteomics insight on pathogenicity

et al. 2014

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BackgroundAspergillus terreus is one of the most harmful filamentous fungal pathogen of humans, animals and plants. Recently, researchers have discovered that A. terreus can cause foliar blight disease in potato (Solanum tuberosum L.). We used light and scanning electron microscopy, and performed proteomics analysis in an attempt to dissect the invasion process of A. terreus in this important crop.ResultsMicroscopic study revealed that invasion of leaf tissue is marked by rapid germination of A. terreus phialidic conidia (PC) by 4 h after inoculation. By 8 h after inoculation, primary germ tubes from PC differentiated into irregular protuberance, often displayed stomata atropism, and failed to penetrate via the epidermal cells. Colonization of leaf tissues was associated with high rate of production of accessory conidia (AC). These analyses showed the occurrence of a unique opposing pattern of AC, tissue-specific and produced on melanized colonizing hyphae during the infection of leaf tissue. A significant proteome change hallmarked by differential expression of class I patatin, lipoxygenase, catalase-peroxidase complex, and cysteine proteinase inhibitor were observed during tuber colonization. These proteins are often involved in signal transduction pathways and crosstalk in pathogenic responses.ConclusionA. terreus abundantly produced AC and multipolar germinating PC to invade potato leaf tissue. Additionally, A. terreus differentially induced enzymes in potato tuber during colonization which facilitates rapid disease development.

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Identification of novel cell wall destabilizing antifungal compounds using a conditional Aspergillus nidulans protein kinase C mutant

Abstract: This work demonstrates the feasibility of using a conditional Aspergillus mutant to conduct a small-molecule library screen to identify novel 'hit' compounds affecting cell wall integrity.

Cited by 12 publications

References 30 publications

The Aspergillus nidulans bimC4 mutation provides an excellent tool for identification of kinesin-14 inhibitors

The Aspergillus nidulans bimC4 mutation provides an excellent tool for identification of kinesin-14 inhibitors

Identification and Characterization of a Novel Family of Selective Antifungal Compounds (CANBEFs) That Interfere with Fungal Protein Synthesis

Invasion of Solanum tuberosum L. by Aspergillus terreus: a microscopic and proteomics insight on pathogenicity

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