Dataset Information

Screening of Pandemic Response Box Library Reveals the High Activity of Olorofim against Pathogenic Sporothrix Species.

ABSTRACT: The increase in the prevalence and severity of fungal infections and the resistance to available antifungals highlights the imperative need for novel therapeutics and the search for new targets. High-content screening of libraries containing hundreds of compounds is a powerful strategy for searching for new drug candidates. In this study, we screened the Pandemic Response Box library (Medicines for Malaria Venture) of 400 diverse molecules against the Sporothrix pathogenic species. The initial screen identified twenty-four candidates that inhibited the growth of Sporothrix brasiliensis by more than 80%. Some of these compounds are known to display antifungal activity, including olorofim (MMV1782354), a new antifungal drug. Olorofim inhibited and killed the yeasts of S. brasiliensis, S. schenckii, and S. globosa at concentrations lower than itraconazole, and it also showed antibiofilm activity. According to the results obtained by fluorimetry, electron microscopy, and particle characterization analyses, we observed that olorofim induced profound alterations on the cell surface and cell cycle arrest in S. brasiliensis yeasts. We also verified that these morphophysiological alterations impaired their ability to adhere to keratinocytes in vitro. Our results indicate that olorofim is a promising new antifungal against sporotrichosis agents.

SUBMITTER: Borba-Santos LP

PROVIDER: S-EPMC9605001 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Screening of Pandemic Response Box Library Reveals the High Activity of Olorofim against Pathogenic <i>Sporothrix</i> Species.

Borba-Santos Luana Pereira LP Rollin-Pinheiro Rodrigo R da Silva Fontes Yasmin Y Dos Santos Giulia Maria Pires GMP de Sousa Araújo Glauber Ribeiro GR Rodrigues Anderson Messias AM Guimarães Allan J AJ de Souza Wanderley W Frases Susana S Ferreira-Pereira Antonio A Barreto-Bergter Eliana E Rozental Sonia S

Journal of fungi (Basel, Switzerland) 20220925 10

The increase in the prevalence and severity of fungal infections and the resistance to available antifungals highlights the imperative need for novel therapeutics and the search for new targets. High-content screening of libraries containing hundreds of compounds is a powerful strategy for searching for new drug candidates. In this study, we screened the Pandemic Response Box library (Medicines for Malaria Venture) of 400 diverse molecules against the <i>Sporothrix</i> pathogenic species. The in ...[more]

PMID: 36294569

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Project description:Scedosporium and Lomentospora species are opportunistic filamentous fungi that cause localized and disseminated infections in immunocompetent and immunocompromised patients. These species are considered resistant fungi due to their low susceptibility to most current antifungal agents used in healthcare settings. The search for new compounds that could work as promising candidate antifungal drugs is an increasing field of interest. In this context, in the present study we screened the Pandemic Response Box® library (Medicines for Malaria Venture [MMV], Switzerland) to identify compounds with antifungal activity against Scedosporium and Lomentospora species. An initial screening of the drugs from this collection at 5 μM was performed using a clinical Scedosporium aurantiacum isolate according to the EUCAST protocol. Compounds with activity against this fungus were also tested against four other species (S. boydii¸ S. dehoogii, S. apiospermum and L. prolificans) at concentrations ranging from 0.078 to 10 μM. Seven compounds inhibited more than 80% of S. aurantiacum growth, three of them (alexidine, amorolfine and olorofim) were selected due to their differences in mechanism of action, especially when compared to drugs from the azole class. These compounds were more active against biofilm formation than against preformed biofilm in Scedosporium and Lomentospora species, except alexidine, which was able to decrease preformed biofilm about 50%. Analysis of the potential synergism of these compounds with voriconazole and caspofungin was performed by the checkerboard method for S. aurantiacum. The analysis by Bliss methodology revealed synergistic effects among selected drugs with caspofungin. When these drugs were combined with voriconazole, only alexidine and amorolfine showed a synergistic effect, whereas olorofim showed an antagonistic effect. Scanning electron microscopy revealed that alexidine induces morphology alterations in S. aurantiacum biofilm grown on a catheter surface. Reactive oxygen species production, mitochondrial activity and surface components were analyzed by fluorescent probes when S. aurantiacum was treated with selected drugs and revealed that some cell parameters are altered by these compounds. In conclusion, alexidine, amorolfine and olorofim were identified as promising compounds to be studied against scedosporiosis and lomentosporiosis.

Project description:BackgroundSporotrichosis is a chronic (sub)cutaneous infection caused by thermodimorphic fungi in the order, Ophiostomatales. These fungi are characterized by major differences in routes of transmission, host predilections, species virulence, and susceptibilities to antifungals. Sporothrix species emerge in the form of outbreaks. Large zoonoses and sapronoses are ongoing in Brazil and China, respectively. Current diagnostic methods based on morphology and physiology are inaccurate due to closely related phenotypes with overlapping components between pathogenic and non-pathogenic Sporothrix. There is a critical need for new diagnostic tools that are specific, sensitive, and cost-effective.MethodologyWe developed a panel of novel markers, based on calmodulin (CAL) gene sequences, for the large-scale diagnosis and epidemiology of clinically relevant members of the Sporothrix genus, and its relative, Ophiostoma. We identified specific PCR-based markers for S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida, and O. stenoceras. We employed a murine model of disseminated sporotrichosis to optimize a PCR assay for detecting Sporothrix in clinical specimens.ResultsPrimer-BLAST searches revealed candidate sequences that were conserved within a single species. Species-specific primers showed no significant homology with human, mouse, or microorganisms outside the Sporothrix genus. The detection limit was 10-100 fg of DNA in a single round of PCR for identifying S. brasiliensis, S. schenckii, S. globosa, S. mexicana, and S. pallida. A simple, direct PCR assay, with conidia as a source of DNA, was effective for rapid, low-cost genotyping. Samples from a murine model of disseminated sporotrichosis confirmed the feasibility of detecting S. brasiliensis and S. schenckii DNA in spleen, liver, lungs, heart, brain, kidney, tail, and feces of infected animals.ConclusionsThis PCR-based method could successfully detect and identify a single species in samples from cultures and from clinical specimens. The method proved to be simple, high throughput, sensitive, and accurate for diagnosing sporotrichosis.

Project description:Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of Sporothrix species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in S. brasiliensis, S. schenckii, and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene pks1, which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU80 homolog that is critical for non-homologous end-joining DNA repair. The use of Δku80 strains from S. brasiliensis enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species. IMPORTANCE Sporotrichosis caused by Sporothrix brasiliensis is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in Sporothrix, this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of Sporothrix species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.

Dataset Information

Screening of Pandemic Response Box Library Reveals the High Activity of Olorofim against Pathogenic Sporothrix Species.

Publications

Screening of Pandemic Response Box Library Reveals the High Activity of Olorofim against Pathogenic <i>Sporothrix</i> Species.

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