High-Throughput Screening of the Repurposing Hub Library to Identify Drugs with Novel Inhibitory Activity against Candida albicans and Candida auris Biofilms.

Candidiasis is one of the most frequent nosocomial infections affecting an increasing number of at-risk patients. Candida albicans remains the most frequent causative agent of candidiasis, but, in the last decade, C. auris has emerged as a formidable multi-drug-resistant pathogen. Both species are fully capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. Here, we have screened the Repurposing Hub library from the Broad Institute, containing over 6000 compounds, in search for inhibitors of C. albicans and C. auris biofilm formation. The primary screen identified 57 initial hits against C. albicans and 33 against C. auris. Confirmatory concentration-dependent assays were used to validate the activity of the initial hits and, at the same time, establish their anti-biofilm potency. Based on these results, ebselen, temsirolimus, and compound BAY 11-7082 emerged as the leading repositionable compounds. Subsequent experiments established their spectrum of antifungal activity against yeasts and filamentous fungi. In addition, their in vivo activity was examined in the murine models of hematogenously disseminated C. albicans and C. auris infections. Although promising, further in vitro and in vivo studies are needed to confirm their potential use for the therapy of candidiasis and possibly other fungal infections.

Development of an Imaging Flow Cytometry Method for Fungal Cytological Profiling and Its Potential Application in Antifungal Drug Development.

Automated imaging techniques have been in increasing demand for the more advanced analysis and efficient characterization of cellular phenotypes. The success of the image-based profiling method hinges on assays that can rapidly and simultaneously capture a wide range of phenotypic features. We have developed an automated image acquisition method for fungal cytological profiling (FCP) using an imaging flow cytometer that can objectively measure over 250 features of a single fungal cell. Fungal cells were labeled with calcofluor white and FM4-64FX, which bind to the cell wall and lipophilic membrane, respectively. Images of single cells were analyzed using IDEAS® software. We first acquired FCPs of fungal cells treated with fluconazole, amphotericin B, and caspofungin, each with a distinct mode of action, to establish FCP databases of profiles associated with specific antifungal treatment. Once fully established, we investigated the potential application of this technique as a screening methodology to identify compounds with novel antifungal activity against Candida albicans and Cryptococcus neoformans. Altogether, we have developed a rapid, powerful, and novel image-profiling method for the phenotypic characterization of fungal cells, also with potential applications in antifungal drug development.

A 384-Well Microtiter Plate Model for Candida Biofilm Formation and Its Application to High-Throughput Screening.

Candidiasis, infections caused by Candida spp., represents one of the most common nosocomial infections afflicting an expanding number of compromised patients. Antifungal therapeutic options are few and show limited efficacy. Moreover, biofilm formation is frequently associated with different manifestations of candidiasis and further complicates therapy. Thus, there is an urgent need for new effective therapeutic agents, particularly those with anti-biofilm activity. Here we describe the development of a novel, simple, fast, economical, and highly reproducible 384-well microtiter plate model for the formation of both Candida albicans and Candida auris biofilms and its application in high-throughput screening (HTS) techniques.

Screening the CALIBR ReFRAME Library in Search for Inhibitors of Candida auris Biofilm Formation.

Candida auris is an emerging yeast which, since its first isolation about a decade ago, has spread rapidly and triggered major infectious outbreaks in health care facilities around the world. C. auris strains often display resistance to clinically-used antifungal agents, contributing to high mortality rates. Thus, there is an urgent need for new antifungals to contain the spread of this emerging multi-drug resistant pathogen and to improve patient outcomes. However, the timeline for the development of a new antifungal agent typically exceeds 10­15 years. Thus, repurposing of current drugs could significantly accelerate the development and eventual deployment of novel therapies for the treatment of C. auris infections. Toward this end, in this study we have profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules in search for known molecules with antifungal activity against C. auris; more specifically, those capable of inhibiting C. auris biofilm formation. From this library, 100 compounds displaying antifungal activity were identified in the initial screen, including 26 compounds for which a dose-response relationship with biofilm-inhibitory activity against C. auris could be confirmed. Of these, five were identified as the most interesting potential repositionable candidates. Due to their known pharmacological and human safety profiles, identification of such compounds should allow for their accelerated preclinical and clinical development for the treatment of C. auris infections.

Current Antimycotics, New Prospects, and Future Approaches to Antifungal Therapy.

Fungal infections represent an increasing threat to a growing number of immune- and medically compromised patients. Fungi are eukaryotic organisms and, as such, there is a limited number of selective targets that can be exploited for antifungal drug development. This has also resulted in a very restricted number of antifungal drugs that are clinically available for the treatment of invasive fungal infections at the present time-polyenes, azoles, echinocandins, and flucytosine. Moreover, the utility of available antifungals is limited by toxicity, drug interactions and the emergence of resistance, which contribute to high morbidity and mortality rates. This review will present a brief summary on the landscape of current antifungals and those at different stages of clinical development. We will also briefly touch upon potential new targets and opportunities for novel antifungal strategies to combat the threat of fungal infections.

Screening Repurposing Libraries for Identification of Drugs with Novel Antifungal Activity.

Fungal organisms are ubiquitous in nature, and progress of modern medicine is creating an expanding number of severely compromised patients susceptible to a variety of opportunistic fungal infections. These infections are difficult to diagnose and treat, leading to high mortality rates. The limited antifungal arsenal, the toxicity of current antifungal drugs, the development of resistance, and the emergence of new multidrug-resistant fungi, all highlight the urgent need for new antifungal agents. Unfortunately, the development of a novel antifungal is a rather long and expensive proposition, and no new classes of antifungal agents have reached the market in the last 2 decades. Drug repurposing, or finding new indications for old drugs, represents a promising alternative pathway to drug development that is particularly appealing within the academic environment. In the last few years, there has been a growing interest in repurposing approaches in the antifungal arena, with multiple groups of investigators having performed screenings of different repurposing libraries against different pathogenic fungi in search for drugs with previously unrecognized antifungal effects. Overall, these repurposing efforts may lead to the fast deployment of drugs with novel antifungal activity, which can rapidly bring benefits to patients, while at the same time reducing health care costs.

Repositionable Compounds with Antifungal Activity against Multidrug Resistant Candida auris Identified in the Medicines for Malaria Venture's Pathogen Box.

Background. Candida auris has spread rapidly around the world as a causative agent of invasive candidiasis in health care facilities and there is an urgent need to find new options for treating this emerging, often multidrug-resistant pathogen. METHODS: We screened the Pathogen Box® chemical library for inhibitors of C. auris strain 0390, both under planktonic and biofilm growing conditions. RESULTS: The primary screen identified 12 compounds that inhibited at least 60% of biofilm formation or planktonic growth. After confirmatory dose-response assays, iodoquinol and miltefosine were selected as the two main leading repositionable compounds. Iodoquinol displayed potent in vitro inhibitory activity against planktonic C. auris but showed negligible inhibitory activity against biofilms; whereas miltefosine was able to inhibit the growth of C. auris under both planktonic and biofilm-growing conditions. Subsequent experiments confirmed their activity against nine other strains C. auris clinical isolates, irrespective of their susceptibility profiles against conventional antifungals. We extended our studies further to seven different species of Candida, also with similar findings. CONCLUSION: Both drugs possess broad spectrum of activity against Candida spp., including multiple strains of the emergent C. auris, and may constitute promising repositionable options for the development of novel therapeutics for the treatment of candidiasis.

Candida albicans biofilm growth and dispersal: contributions to pathogenesis.

The fungal species Candida albicans is most frequently associated with biofilm formation in immune-compromised and medically compromised patients, and it is now firmly established that biofilm formation represents a major virulence factor during candidiasis. A growing body of evidence has demonstrated that C. albicans biofilm development is a highly regulated and coordinated process, where adhesive interactions, morphogenetic conversions, and consortial behavior play significant roles. Cells within the biofilms are protected from environmental stresses including host immune defenses and antifungal treatment, which carries important clinical consequences for the treatment of biofilm-associated infections. Dispersal of cells from biofilms represents one of the hallmarks of the biofilm life-style, and in the case of C. albicans dispersed cells are responsible for candidemia and dissemination leading to the establishment of invasive disease.

Screening a Repurposing Library for Inhibitors of Multidrug-Resistant Candida auris Identifies Ebselen as a Repositionable Candidate for Antifungal Drug Development.

Since its original isolation in 2009, Candida auris has spread across the globe as a causative agent of invasive candidiasis. C. auris is typically intrinsically resistant to fluconazole and can also be resistant to echinocandins and even amphotericin B. Thus, there is an urgent need to find new treatment options against this emerging pathogen. To address this growing problem, we performed a screen of the Prestwick Chemical library, a repurposing library of 1,280 small molecules, consisting mostly of approved off-patent drugs, in search of those with activity against a multidrug-resistant C. auris isolate. Our initial screen, using standardized susceptibility testing methodologies, identified nine miscellaneous compounds with no previous clinical indication as antifungals or antiseptics that displayed activity against C. auris Confirmation and follow-up studies identified ebselen as the drug displaying the most potent activity, with 100% inhibition of growth detected at concentrations as low as 2.5 µM. We further evaluated the ability of ebselen to inhibit C. auris biofilm formation and examined the effects of combination therapies of ebselen with clinically used antifungals. We extended our studies to different C. auris strains with various susceptibility patterns and also confirmed its antifungal activity against Candida albicans and clinical isolates of multiple other Candida species. Furthermore, ebselen displayed a broad spectrum of antifungal actions on the basis of its activity against a variety of medically important fungi, including yeasts and molds. Overall, our results indicate the promise of ebselen as a repositionable agent for the treatment of candidiasis and possibly other mycoses and, in particular, for the treatment of infections refractory to conventional treatment with current antifungals.

Systematic Complex Haploinsufficiency-Based Genetic Analysis of Candida albicans Transcription Factors: Tools and Applications to Virulence-Associated Phenotypes.

Genetic interaction analysis is a powerful approach to the study of complex biological processes that are dependent on multiple genes. Because of the largely diploid nature of the human fungal pathogen Candida albicans, genetic interaction analysis has been limited to a small number of large-scale screens and a handful for gene-by-gene studies. Complex haploinsufficiency, which occurs when a strain containing two heterozygous mutations at distinct loci shows a phenotype that is distinct from either of the corresponding single heterozygous mutants, is an expedient approach to genetic interactions analysis in diploid organisms. Here, we describe the construction of a barcoded-library of 133 heterozygous TF deletion mutants and deletion cassettes for designed to facilitate complex haploinsufficiency-based genetic interaction studies of the TF networks in C. albicans We have characterized the phenotypes of these heterozygous mutants under a broad range of in vitro conditions using both agar-plate and pooled signature tag-based assays. Consistent with previous studies, haploinsufficiency is relative uncommon. In contrast, a set of 12 TFs enriched in mutants with a role in adhesion were found to have altered competitive fitness at early time points in a murine model of disseminated candidiasis. Finally, we characterized the genetic interactions of a set of biofilm related TFs in the first two steps of biofilm formation, adherence and filamentation of adherent cells. The genetic interaction networks at each stage of biofilm formation are significantly different indicating that the network is not static but dynamic.

The Candida albicans Biofilm Matrix: Composition, Structure and Function.

A majority of infections caused by Candida albicans-the most frequent fungal pathogen-are associated with biofilm formation. A salient feature of C. albicans biofilms is the presence of the biofilm matrix. This matrix is composed of exopolymeric materials secreted by sessile cells within the biofilm, in which all classes of macromolecules are represented, and provides protection against environmental challenges. In this review, we summarize the knowledge accumulated during the last two decades on the composition, structure, and function of the C. albicans biofilm matrix. Knowledge of the matrix components, its structure, and function will help pave the way to novel strategies to combat C. albicans biofilm infections.