Development of Anti-Virulence Approaches for Candidiasis via a Novel Series of Small-Molecule Inhibitors of Candida albicans Filamentation

Abstract

Candida albicans remains the main etiologic agent of candidiasis, the most common fungal infection and now the third most frequent infection in U.S. hospitals. The scarcity of antifungal agents and their limited efficacy contribute to the unacceptably high morbidity and mortality rates associated with these infections. The yeast-to-hypha transition represents the main virulence factor associated with the pathogenesis of C. albicans infections. In addition, filamentation is pivotal for robust biofilm development, which represents another major virulence factor for candidiasis and further complicates treatment. Targeting pathogenic mechanisms rather than growth represents an attractive yet clinically unexploited approach in the development of novel antifungal agents. Here, we performed large-scale phenotypic screening assays with 30,000 drug-like small-molecule compounds within ChemBridge's DIVERSet chemical library in order to identify small-molecule inhibitors of C. albicans filamentation, and our efforts led to the identification of a novel series of bioactive compounds with a common biaryl amide core structure. The leading compound of this series, N-[3-(allyloxy)-phenyl]-4-methoxybenzamide, was able to prevent filamentation under all liquid and solid medium conditions tested, suggesting that it impacts a common core component of the cellular machinery that mediates hypha formation under different environmental conditions. In addition to filamentation, this compound also inhibited C. albicans biofilm formation. This leading compound also demonstrated in vivo activity in clinically relevant murine models of invasive and oral candidiasis. Overall, our results indicate that compounds within this series represent promising candidates for the development of novel anti-virulence approaches to combat C. albicans infections.IMPORTANCE Since fungi are eukaryotes, there is a limited number of fungus-specific targets and, as a result, the antifungal arsenal is exceedingly small. Furthermore, the efficacy of antifungal treatment is compromised by toxicity and development of resistance. As a consequence, fungal infections carry high morbidity and mortality rates, and there is an urgent but unmet need for novel antifungal agents. One appealing strategy for antifungal drug development is to target pathogenetic mechanisms associated with infection. In Candida albicans, one of the most common pathogenic fungi, morphogenetic transitions between yeast cells and filamentous hyphae represent a key virulence factor associated with the ability of fungal cells to invade tissues, cause damage, and form biofilms. Here, we describe and characterize a novel small-molecule compound capable of inhibiting C. albicans filamentation both in vitro and in vivo; as such, this compound represents a leading candidate for the development of anti-virulence therapies against candidiasis.

Keywords: Candida albicans; anti-virulence factor; antifungal drugs; filamentation; large-scale phenotypic screening.

FIG 1

Primary screen to identify inhibitors of C. albicans filamentation. (A) Schematic diagram of the phenotypic assay used for large-scale phenotypic screening of 30,000 small-molecule compounds in the DIVERSet chemical library, which we used in our search for inhibitors of C. albicans filamentation. The screening uses 96-well round-bottom microtiter plates and takes advantage of tight control via doxycycline of morphogenetic conversions in the C. albicans tet-NRG1 strain. Individual wells of the microtiter plates are seeded with fungal cells in the presence of 5 µM each individual compound, with appropriate positive and negative controls. The plates are incubated at 37°C and visually inspected at 2 h, 4 h, and 24 h. Under the conditions used, wells containing cells that grow the filamentous form (uninhibited by the presence of the compound) appear cloudy, whereas cells that grow in the yeast form (due to inhibition of filamentation in the presence of a hit compound) fall to the bottom of the wells and form rings, which are easily discernible macroscopically. Microscopy is then used for confirmation of the inhibitory effect on filamentation. (B) Chemical structures of compounds 9029936 and 7977044, two of the major initial hits identified in the primary screen. The two compounds share a common biaryl amide motif (highlighted in red). (C) Identity, physicochemical properties, including the clogP (partition coefficient and a measure of lipophilicity), tPSA (molecular polar surface area), and logSw (solubility of the drug in water), as well as IC₅₀ (potency) and CC₅₀ (toxicity) values, for these two small-molecule compounds.

FIG 2

Inhibitory effects of the leading compounds on C. albicans filamentation. (A) Photomicrographs showing morphology of C. albicans strain SC5314 grown for 6 h under strong filament-inducing conditions (YPD plus serum, 37°C) in the presence of compounds 9029936 and 7977044. (B) Inhibition of filamentation in different liquid media. Liquid cultures of C. albicans strain SC5314 were grown in a variety of hypha-inducing media in the absence or presence of compound 9029936 at a concentration of 5 μM. Aliquots were taken from the different cultures at 6 h postinduction, visualized by DIC microscopy, and photographed. Bars, 20 μm.

FIG 3

Inhibitory effects of leading compound 9029936 on C. albicans filamentation on solid media. (A) Colony morphologies of C. albicans strain SC5314 grown on various solid filament-inducing media in the absence or presence of compound 9029936 at 5 μM. Plates were incubated at 37°C for 5 days. A wrinkled colony morphology indicates the presence of filamentous cells. (B) Compound 9029936 inhibits C. albicans agar invasion. C. albicans SC5314 cells were streaked on YPD plates with or without compound 9029936 at 5 μM, and the plates were incubated at 37°C for 5 days. The plates were photographed prior to (left) and after (right) surface cells were removed by gentle washing under running water. In the presence of the compound, cells grew mostly on the surface of the medium and were washed away, whereas cells grown in the absence of the compound had invaded the agar and remained after washing.

FIG 4

Compounds 9029936 and 7977044 inhibit C. albicans filamentation that is driven by overexpression of BRG1. In the absence of doxycycline, overexpression of BRG1 in the C. albicans tet-BRG1 strain led to filamentation under noninducing conditions (YPD medium at 30°C, no serum). Addition of compound 9029936 or 7977044 led to inhibition of filamentation of this strain under these conditions.

FIG 5

In vitro characterization of the inhibitory activity of the leading compound 9029936 on C. albicans biofilm formation. (A) Dose-dependent inhibitory effects of compound 9029936 on C. albicans biofilm formation. The compound was tested in serial 2-fold dilutions (concentrations ranging from 40 to 0.078 µM), with appropriate positive and negative controls. Results shown are the mean percent biofilm inhibition relative to control biofilms (grown in the absence of compound 9029936), determined in XTT colorimetric assays for multiple technical replicates from several independent experiments. Error bars indicate standard deviations. (B) The leading compound inhibited the proliferation and maturation phases of C. albicans biofilm development. A biofilm kinetic assay was performed in order to examine the inhibitory effects of compound 9029936 at different stages of biofilm development, using the same 96-well microtiter plate model of C. albicans biofilm formation. The extent of inhibition was determined at multiple times (2, 6, 8, 12, 24, and 48 h) after seeding the wells with C. albicans cells in the presence or absence of compound 9029936 (5 μM concentration). Results are expressed as means of multiple technical replicates from a single experiment, with error bars representing standard deviations. (C) SEM images of biofilms of C. albicans strain SC5314 formed in the absence or presence of compound 9029936 at a concentration of 5 μM. Bars, 20 μm.

FIG 6

In vivo activity of the leading compound 9029936 in the clinically relevant murine models of hematogenously disseminated invasive candidiasis and oropharyngeal candidiasis. (A) Protection in the murine model of hematogenously disseminated invasive candidiasis. Survival curves show results for a group of mice treated with compound 9029936 compared to an untreated control group. By the end of the experiment, 87.5% of animals that received treatment with compound 9029936 survived the infection, resulting in statistically significant differences versus the control group. (B) Histological analyses of kidney sections retrieved from untreated mice 3 days after intravenous infection with C. albicans strain SC5314 showed typical kidney lesions of a principally filamentous nature, whereas isolated cells or groups of cells with a mostly yeast morphology were predominant in kidneys recovered from mice treated with compound 9029936 at the same time point. Bars, 20 μm. (C) Efficacy of compound 9029936 against oral candidiasis in mice, as assessed by clinical score. Mice were immunosuppressed and infected orally with C. albicans strain SC5314. One group of mice received vehicle only and served as untreated controls, while another group of mice received treatment with compound 9029936. Mice were monitored for signs of oral candidiasis and assigned a clinical score on the basis of the extent and severity of tongue lesions. The graph depicts clinical scores for mice sacrificed 3 days after infection, for which there were statistically significant differences between treated and control mice. (D) Histological analyses revealed an abundant biofilm composed mostly of filamentous cells covering the tongue surface and penetrating deep into the tissues of untreated animals, whereas fewer cells, displaying mostly an elongated yeast morphology and superficially located, were detected in tongues from mice treated with the leading compound 9029936. Bars, 20 μm.