Unraveling the Biology of a Fungal Meningitis Pathogen Using Chemical Genetics

Brown, Jessica C. S.; Nelson, J. Daniel; VanderSluis, Benjamin; Deshpande, Raamesh; Butts, Arielle; Kagan, Sarah; Polacheck, Itzhack; Krysan, Damian J.; Myers, Chad L.; Madhani, Hiten D.

doi:10.1016/j.cell.2014.10.044

Cited by 71 publications

(92 citation statements)

References 63 publications

(107 reference statements)

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“…The echinocandins are a class of cyclic hexapeptides with a linoleoyl side chain, targeting glucans of fungal cell walls [38]. The over-reliance on limited drug targets has led to the rapid emergence of anti-Candida drug resistance [39]. Thus, it is imperative to identify new anti-Candida drugs with distinct chemical structures and novel mode of actions.…”

Section: Discussionmentioning

confidence: 99%

Alteramide B is a microtubule antagonist of inhibiting Candida albicans

Ding

et al. 2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Alteramide B (ATB), isolated from Lysobacter enzymogenes C3, was a new polycyclic tetramate macrolactam (PTM). ATB exhibited potent inhibitory activity against several yeasts, particularly Candida albicans SC5314, but its antifungal mechanism is unknown. Methods The structure of ATB was established by extensive spectroscopic analyses, including high-resolution mass spectrometry, 1D- and 2D-NMR, and CD spectra. Flow cytometry, fluorescence microscope, transmission electron microscope, molecular modeling, overexpression and site-directed mutation studies were employed to delineate the anti-candida molecular mechanism of ATB. Results ATB induced apoptosis in C. albicans through inducing reactive oxygen species (ROS) production by disrupting microtubules. Molecular dynamics studies revealed the binding patterns of ATB to the β-tubulin subunit. Overexpression of the wild type and site-directed mutants of the β-tubulin gene (TUBB) changed the sensitivity of C. albicans to ATB, confirming the binding of ATB to β-tubulin, and indicating that the binding sites are L215, L217, L273, L274 and R282. In vivo, ATB significantly improved the survival of the candidiasis mice and reduced fungal burden. Conclusion The molecular mechanism underlying the ATB-induced apoptosis in C. albicans is through inhibiting tubulin polymerization that leads to cell cycle arrest at the G2/M phase. The identification of ATB and the study of its activity provide novel mechanistic insights into the mode of action of PTMs against the human pathogen. General significance This study shows that ATB is a new microtubule inhibitor and a promising anti-Candida lead compound. The results also support β-tubulin as a potential target for anti-Candida drug discovery.

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Section: Discussionmentioning

confidence: 99%

Alteramide B is a microtubule antagonist of inhibiting Candida albicans

Ding

et al. 2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…This was indeed the case, even when the known synergistic molecules have different mechanisms of action [18]. When we validated O2M on the pathogenic fungus Cryptococcus neoformans , we identified 36 new synergistic interactions with a low false positive rate (73% of the predictions were verified) [18]. Since then, several other groups published methods using chemical-genetics datasets to identify synergistic small-molecule interactions [19–21].…”

Section: Introductionmentioning

confidence: 99%

High-throughput identification and rational design of synergistic small-molecule pairs for combating and bypassing antibiotic resistance

et al. 2017

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Antibiotic-resistant infections kill approximately 23,000 people and cost $20,000,000,000 each year in the United States alone despite the widespread use of small-molecule antimicrobial combination therapy. Antibiotic combinations typically have an additive effect: the efficacy of the combination matches the sum of the efficacies of each antibiotic when used alone. Small molecules can also act synergistically when the efficacy of the combination is greater than the additive efficacy. However, synergistic combinations are rare and have been historically difficult to identify. High-throughput identification of synergistic pairs is limited by the scale of potential combinations: a modest collection of 1,000 small molecules involves 1 million pairwise combinations. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, the overlap2 method (O2M). O2M extracts patterns from chemical-genetic datasets, which are created when a collection of mutants is grown in the presence of hundreds of different small molecules, producing a precise set of phenotypes induced by each small molecule across the mutant set. The identification of mutants that show the same phenotype when treated with known synergistic molecules allows us to pinpoint additional molecule combinations that also act synergistically. As a proof of concept, we focus on combinations with the antibiotics trimethoprim and sulfamethizole, which had been standard treatment against urinary tract infections until widespread resistance decreased efficacy. Using O2M, we screened a library of 2,000 small molecules and identified several that synergize with the antibiotic trimethoprim and/or sulfamethizole. The most potent of these synergistic interactions is with the antiviral drug azidothymidine (AZT). We then demonstrate that understanding the molecular mechanism underlying small-molecule synergistic interactions allows the rational design of additional combinations that bypass drug resistance. Trimethoprim and sulfamethizole are both folate biosynthesis inhibitors. We find that this activity disrupts nucleotide homeostasis, which blocks DNA replication in the presence of AZT. Building on these data, we show that other small molecules that disrupt nucleotide homeostasis through other mechanisms (hydroxyurea and floxuridine) also act synergistically with AZT. These novel combinations inhibit the growth and virulence of trimethoprim-resistant clinical Escherichia coli and Klebsiella pneumoniae isolates, suggesting that they may be able to be rapidly advanced into clinical use. In sum, we present a generalizable method to screen for novel synergistic combinations, to identify particular mechanisms resulting in synergy, and to use the mechanistic knowledge to rationally design new combinations that bypass drug resistance.

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“…3 However, Candida species has been reported to show azole resistances through upregulation of efflux, mutation and overexpression to confront the variety of stress conditions. 4, 5 New anti-candida drugs with distinct chemical structures and mechanism of action are urgently required.…”

Section: Introductionmentioning

confidence: 99%

HSAF-induced antifungal effects in Candida albicans through ROS-mediated apoptosis

Ding

et al. 2016

RSC Adv.

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Heat-stable antifungal factor (HSAF) belongs to polycyclic tetramate macrolactams (PTMs), which inhibits many fungal pathogens and is effective in inhibiting Candida albicans (C. albicans). In this study, we found that HSAF induced the apoptosis of Candida albicans SC5314 through inducing the production of reactive oxygen species (ROS). Nevertheless, we validated the efficacy of HSAF against candidiasis caused by C. albicans in a murine model in vivo, and HSAF significantly improved survival and reduced fungal burden compared to vehicles. A molecular dynamics (MD) simulation was also investigated, revealing the theoretical binding mode of HSAF to the β-tubulin of C. albicans. This study first found PTMs-induced fungal apoptosis through ROS accumulation in C. albicans and its potential as a novel agent for fungicides.

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Unraveling the Biology of a Fungal Meningitis Pathogen Using Chemical Genetics

Cited by 71 publications

References 63 publications

Alteramide B is a microtubule antagonist of inhibiting Candida albicans

Alteramide B is a microtubule antagonist of inhibiting Candida albicans

High-throughput identification and rational design of synergistic small-molecule pairs for combating and bypassing antibiotic resistance

HSAF-induced antifungal effects in Candida albicans through ROS-mediated apoptosis

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