Derivatives of the Antimalarial Drug Mefloquine Are Broad-Spectrum Antifungal Molecules with Activity against Drug-Resistant Clinical Isolates

ABSTRACT The antifungal pharmacopeia is critically small, particularly in light of the recent emergence of multidrug-resistant pathogens, such as Candida auris. Here, we report that derivatives of the antimalarial drug mefloquine have broad-spectrum antifungal activity against pathogenic yeasts and molds. In addition, the mefloquine derivatives have activity against clinical isolates that are resistant to one or more of the three classes of antifungal drugs currently used to treat invasive fungal infections, indicating that they have a novel mechanism of action. Importantly, the in vitro toxicity profiles obtained using human cell lines indicated that the toxicity profiles of the mefloquine derivatives are very similar to those of the parent mefloquine, despite being up to 64-fold more active against fungal cells. In addition to direct antifungal activity, subinhibitory concentrations of the mefloquine derivatives inhibited the expression of virulence traits, including filamentation in Candida albicans and capsule formation/melanization in Cryptococcus neoformans. Mode/mechanism-of-action experiments indicated that the mefloquine derivatives interfere with both mitochondrial and vacuolar function as part of a multitarget mechanism of action. The broad-spectrum scope of activity, blood-brain barrier penetration, and large number of previously synthesized analogs available combine to support the further optimization and development of the antifungal activity of this general class of drug-like molecules.

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Derivatives of the antimalarial drug mefloquine are broad spectrum antifungal molecules with activity against drug-resistant clinical isolates

10.1101/851816 ◽

2019 ◽

Author(s):

Marhiah C. Montoya ◽

Sarah Beattie ◽

Kathryn M. Alden ◽

Damian J. Krysan

Keyword(s):

Antifungal Activity ◽

Mechanism Of Action ◽

Broad Spectrum ◽

Fungal Infections ◽

Clinical Isolates ◽

In Vitro Toxicity ◽

Drug Resistant ◽

Candida Auris ◽

Recent Emergence ◽

Derivatives Of

ABSTRACTThe antifungal pharmacopeia is critically small, particularly in light of the recent emergence of multi-drug-resistant pathogens such as Candida auris. Herein, we report that derivatives of the anti-malarial drug mefloquine have broad spectrum antifungal activity against pathogenic yeasts and molds. In addition, the mefloquine derivatives have activity against clinical isolates that are resistant to one or more of the three classes of drugs currently used to treat invasive fungal infections, indicating that they have a novel mechanism of action. Importantly, the in vitro toxicity profiles using human cell lines indicate that the mefloquine derivatives are very similar to the parent mefloquine despite being up to 64-fold more active against fungal cells. In addition to direct antifungal activity, sub-inhibitory concentrations of the mefloquine derivatives inhibit the expression of virulence traits including filamentation in C. albicans and capsule formation/melanization in C. neoformans. Mode/mechanism of action experiments indicate that the mefloquine derivatives interfere with both mitochondrial and vacuolar function as part of a multi-target mechanism of action. The broad-spectrum scope of activity, blood-brain-barrier penetration, and large number of previously synthesized analogs available combine to support the further optimization and development of the antifungal activity of this general class of drug-like molecules.

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Crystal Structure of the New Investigational Drug Candidate VT-1598 in Complex with Aspergillus fumigatus Sterol 14α-Demethylase Provides Insights into Its Broad-Spectrum Antifungal Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00570-17 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 29

Author(s):

Tatiana Y. Hargrove ◽

Edward P. Garvey ◽

William J. Hoekstra ◽

Christopher M. Yates ◽

Zdzislaw Wawrzak ◽

...

Keyword(s):

Antifungal Activity ◽

Aspergillus Fumigatus ◽

Broad Spectrum ◽

Fungal Infections ◽

Enzymatic Reaction ◽

Heme Iron ◽

Antifungal Drugs ◽

Drug Candidate ◽

Investigational Drug ◽

Content Type

ABSTRACT Within the past few decades, the incidence and complexity of human fungal infections have increased, and therefore, the need for safer and more efficient, broad-spectrum antifungal agents is high. In the study described here, we characterized the new tetrazole-based drug candidate VT-1598 as an inhibitor of sterol 14α-demethylase (CYP51B) from the filamentous fungus Aspergillus fumigatus. VT-1598 displayed a high affinity of binding to the enzyme in solution (dissociation constant, 13 ± 1 nM) and in the reconstituted enzymatic reaction was revealed to have an inhibitory potency stronger than the potencies of all other simultaneously tested antifungal drugs, including fluconazole, voriconazole, ketoconazole, and posaconazole. The X-ray structure of the VT-1598/A. fumigatus CYP51 complex was determined and depicts the distinctive binding mode of the inhibitor in the enzyme active site, suggesting the molecular basis of the improved drug potency and broad-spectrum antifungal activity. These data show the formation of an optimized hydrogen bond between the phenoxymethyl oxygen of VT-1598 and the imidazole ring nitrogen of His374, the CYP51 residue that is highly conserved across fungal pathogens and fungus specific. Comparative structural analysis of A. fumigatus CYP51/voriconazole and Candida albicans CYP51/VT-1161 complexes supports the role of H bonding in fungal CYP51/inhibitor complexes and emphasizes the importance of an optimal distance between this interaction and the inhibitor-heme iron interaction. Cellular experiments using two A. fumigatus strains (strains 32820 and 1022) displayed a direct correlation between the effects of the drugs on CYP51B activity and fungal growth inhibition, indicating the noteworthy anti-A. fumigatus potency of VT-1598 and confirming its promise as a broad-spectrum antifungal agent.

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Whole-Genome Approach to Understanding the Mechanism of Action of a Histatin 5-Derived Peptide

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01698-19 ◽

2019 ◽

Vol 64 (3) ◽

Author(s):

Cody B. Bullock ◽

David S. McNabb ◽

Inés Pinto

Keyword(s):

Mechanism Of Action ◽

Fungal Infections ◽

Antifungal Drugs ◽

Content Type ◽

Histatin 5 ◽

Amino Acid Peptide ◽

Killing Activity ◽

Transport Chain ◽

Mitochondrial Functions ◽

Increased Sensitivity

ABSTRACT The incidence of opportunistic fungal infections that threaten immunocompromised patients, along with the limited arsenal of antifungal drugs, calls for renewed efforts to develop novel antifungal therapies. Antimicrobial peptides have garnered interest as potential therapeutics. Among naturally occurring peptides, histatin 5 is a well-characterized 24-amino-acid peptide with strong antifungal activity. Our lab has identified a smaller histatin derivative, KM29, with stronger activity against multiple Candida spp., prompting us to investigate its fungicidal mechanism. A genetic screen was developed to test the Saccharomyces cerevisiae genomewide deletion collection for mutants with increased or decreased peptide sensitivity. The goal was to identify genes that would reveal insights into the mechanism of action of KM29, to be assessed in Candida albicans. Several biological processes yielded increased sensitivity, with endosomal transport and vacuolar function appearing at high frequencies. Among the pathways involved in increased resistance, mitochondrial function showed the highest normalized genome frequency; hence, we focused on characterizing this pathway. KM29 localizes to mitochondria, and the killing activity depends on a functional electron transport chain. In addition, KM29 triggered reactive oxygen species (ROS) production, which was responsible for some cell death but insufficient to account for the complete killing activity. In agreement with this finding, we found that KM29 induced mitochondrial fragmentation and a mild loss of mitochondrial membrane potential. Furthermore, respiratory mutants exhibited severely diminished KM29 uptake. We confirmed this behavior in a C. albicans respiratory mutant. Taking our findings together, this work delineates the mitochondrial functions associated with KM29 fungicidal activity and provides additional pathways for further characterization in Candida spp.

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Non-toxic Cobalt(III) Schiff Base Complexes with Broad Spectrum Antifungal Activity

10.26434/chemrxiv.12736505 ◽

2020 ◽

Author(s):

Angelo Frei ◽

A. Paden King ◽

Gabrielle J. Lowe ◽

Amy K. Cain ◽

Francesca L. Short ◽

...

Keyword(s):

Schiff Base ◽

Antifungal Activity ◽

Broad Spectrum ◽

Bacterial Infections ◽

Fungal Infections ◽

Antifungal Drugs ◽

New Drugs ◽

Schiff Base Complexes ◽

In Vivo Studies

Resistance to currently available antifungal drugs has quietly been on the rise but overshadowed by the alarming spread of antibacterial resistance. There is a striking lack of attention to the threat of drug resistant fungal infections, with only a handful of new drugs currently in development. Given that metal complexes have proven to be useful new chemotypes in the fight against diseases such as cancer, malaria, and bacterial infections, it stands to reason to explore their possible utility in treating fungal infections. Herein we report a series of cobalt(III) Schiff base complexes with broad spectrum antifungal activity. Some of these complexes (1-3) show minimum inhibitory concentrations (MIC) in the low micro- to nanomolar range against a series of Candida and Cryptococcus yeasts. Additionally, we demonstrate that these compounds show no cytotoxicity against both bacterial and human cells. Finally, we report first in vivo toxicity data on these compounds in Galleria mellonella, showing that doses as high as 266 mg/kg are tolerated without adverse effects, paving the way for further in vivo studies of these complexes. <br>

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Screening of Chemical Libraries for New Antifungal Drugs against Aspergillus fumigatus Reveals Sphingolipids Are Involved in the Mechanism of Action of Miltefosine

mBio ◽

10.1128/mbio.01458-21 ◽

2021 ◽

Author(s):

Thaila Fernanda dos Reis ◽

Maria Augusta Crivelente Horta ◽

Ana Cristina Colabardini ◽

Caroline Mota Fernandes ◽

Lilian Pereira Silva ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Mechanism Of Action ◽

Filamentous Fungus ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Content Type ◽

Chemical Libraries

The filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis, and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over recent decades.

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Toxoflavin Produced byBurkholderia gladiolifromLycoris aureaIs a New Broad-Spectrum Fungicide

Applied and Environmental Microbiology ◽

10.1128/aem.00106-19 ◽

2019 ◽

Vol 85 (9) ◽

Cited By ~ 7

Author(s):

Xiaodan Li ◽

Yikui Li ◽

Ren Wang ◽

Qizhi Wang ◽

Ling Lu

Keyword(s):

Antifungal Activity ◽

Broad Spectrum ◽

Fungal Pathogens ◽

Antifungal Agents ◽

Fungal Infections ◽

Endophytic Bacterium ◽

Content Type ◽

Burkholderia Gladioli ◽

Lycoris Aurea ◽

Resistant Mutants

ABSTRACTFungal infections not only cause extensive agricultural damage but also result in serious diseases in the immunodeficient populations of human beings. Moreover, the increasing emergence of drug resistance has led to a decrease in the efficacy of current antifungals. Thus, screening of new antifungal agents is imperative in the fight against antifungal drug resistance. In this study, we show that an endophytic bacterium,Burkholderia gladioliHDXY-02, isolated from the medicinal plantLycoris aurea, showed broad-spectrum antifungal activity against plant and human fungal pathogens. An antifungal ability assay indicated that the bioactive component was produced from strain HDXY-02 having an extracellular secreted component with a molecular weight lower than 1,000 Da. In addition, we found that this new antifungal could be produced effectively by liquid fermentation of HDXY-02. Furthermore, the purified component contributing to the antifungal activity was identified to be toxoflavin, a yellow compound possessing a pyrimido[5,4-e][1,2,4]triazine ring.In vitrobioactivity studies demonstrated that purified toxoflavin fromB. gladioliHDXY-02 cultures had a significant antifungal activity against the human fungal pathogenAspergillus fumigatus, resulting in abolished germination of conidia. More importantly, the growth inhibition by toxoflavin was observed in both wild-type and drug-resistant mutants (cyp51Aand non-cyp51A) ofA. fumigatus. Finally, an optimized protocol for the large-scale production of toxoflavin (1,533 mg/liter) has been developed. Taken together, our findings provide a promising biosynthetic resource for producing a new antifungal reagent, toxoflavin, from isolates of the endophytic bacteriumB. gladioli.IMPORTANCEHuman fungal infections are a growing problem associated with increased morbidity and mortality. Moreover, a growing number of antifungal-resistant fungal isolates have been reported over the past decade. Thus, the need for novel antifungal agents is imperative. In this study, we show that an endophytic bacterium,Burkholderia gladioli, isolated from the medicinal plantLycoris aurea, is able to abundantly secrete a compound, toxoflavin, which has a strong fungicidal activity not only against plant fungal pathogens but also against human fungal pathogensAspergillus fumigatusandCandida albicans,Cryptococcus neoformans, and the model filamentous fungusAspergillus nidulans. More importantly, toxoflavin also displays an efficacious inhibitory effect against azole antifungal-resistant mutants ofA. fumigatus. Consequently, our findings provide a promising approach to abundantly produce toxoflavin, which has novel broad-spectrum antifungal activity, especially against those currently problematic drug-resistant isolates.

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Antifungal In Vitro Activity of Pilosulin- and Ponericin-Like Peptides from the Giant Ant Dinoponera quadriceps and Synergistic Effects with Antimycotic Drugs

Antibiotics ◽

10.3390/antibiotics9060354 ◽

2020 ◽

Vol 9 (6) ◽

pp. 354 ◽

Cited By ~ 2

Author(s):

Hilania Valéria Dodou Lima ◽

Carolina Sidrim de Paula Cavalcante ◽

Gandhi Rádis-Baptista

Keyword(s):

Antifungal Activity ◽

Antimicrobial Peptides ◽

Broad Spectrum ◽

Culture Medium ◽

Fungal Infections ◽

Synergistic Effects ◽

Antifungal Drugs ◽

Clinical Strain ◽

Onset Of Action

Venoms from ants comprise a rich source of bioactive peptides, including antimicrobial peptides. From the proteome and peptidome of the giant ant Dinoponera quadriceps venom, members of five known classes of antimicrobial peptides were disclosed (e.g., dermaseptin-, defensin-, ICK-, pilosulin- and ponericin-like types). Based on comparative analysis, these family members have structural determinants that indicate they could display antimicrobial activities. In previous works, pilosulin- and ponericin-like peptides were demonstrated to be active against bacteria, fungi, and parasites. Herein, the antifungal activity of ponericin- and pilosulin-like peptides were assessed, aiming at the expansion of the knowledge about AMPs in predatory ants and the development of new microbicide strategies to deal with difficult-to-treat fungal infections. Synthetic pilosulin- (Dq-2562, Dq-1503, and Dq-1319) and ponericin-like (Dq-3162) peptides were evaluated for their fungicide and fungistatic activities against different species of Candida, including a drug-resistant clinical strain. The MICs and MLCs were determined for all peptides individually and in combination with general antifungal drugs by the microdilution method. The time-kill kinetic curves were set up by means of a luminescent reagent, of which the light signal is proportional to the number of viable cells. The candicidal synergism observed by the combination of subinhibitory concentrations of peptides and general antimycotic drugs were quantified by the checkerboard test and fluorescent dye permeation assay. The influence of ergosterol on the antifungal activity was verified by supplementation of culture medium. The pilosulin- (Dq-2562 and Dq-1503) and ponericin-like (Dq-3162) were the most active peptides, displaying a broad spectrum of antifungal activity in vitro, with MICs in the range of 0.625 to 10 µM. The combination of peptides and conventional antimycotic drugs displayed a synergistic reduction in the MIC values of individual peptides and drugs, while soluble ergosterol in the culture medium increased the MICs. The fungicide and fungistatic activity of the individual peptides and peptides in combination with antimycotics were time-dependent with a rapid onset of action and long-lasting effect, which involved membrane disruption as an underlying mechanism of their action. Altogether, pilosulin- and ponericin-like peptides from the giant ant D. quadriceps venom display a broad-spectrum of candicidal activity, what allows their inclusion in the row of the antifungal peptides and gives support for further studies on the development of strategies to fight candidiasis.

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Discovery of Cercosporamide, a Known Antifungal Natural Product, as a Selective Pkc1 Kinase Inhibitor through High-Throughput Screening

Eukaryotic Cell ◽

10.1128/ec.3.4.932-943.2004 ◽

2004 ◽

Vol 3 (4) ◽

pp. 932-943 ◽

Cited By ~ 63

Author(s):

Andrea Sussman ◽

Karen Huss ◽

Li-Chun Chio ◽

Steve Heidler ◽

Margaret Shaw ◽

...

Keyword(s):

Cell Wall ◽

Antifungal Activity ◽

High Throughput ◽

Broad Spectrum ◽

High Throughput Screening ◽

Kinase Inhibitor ◽

Fungal Infections ◽

Antifungal Drugs ◽

Antifungal Compound ◽

Glucan Synthase

ABSTRACT The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broad-spectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a β-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and β-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

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Non-toxic Cobalt(III) Schiff Base Complexes with Broad Spectrum Antifungal Activity

10.26434/chemrxiv.12736505.v1 ◽

2020 ◽

Author(s):

Angelo Frei ◽

A. Paden King ◽

Gabrielle J. Lowe ◽

Amy K. Cain ◽

Francesca L. Short ◽

...

Keyword(s):

Schiff Base ◽

Antifungal Activity ◽

Broad Spectrum ◽

Bacterial Infections ◽

Fungal Infections ◽

Antifungal Drugs ◽

New Drugs ◽

Schiff Base Complexes ◽

In Vivo Studies

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High-Throughput Screening of a Collection of Known Pharmacologically Active Small Compounds for Identification of Candida albicans Biofilm Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00680-13 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3681-3687 ◽

Cited By ~ 62

Author(s):

Samuel A. Siles ◽

Anand Srinivasan ◽

Christopher G. Pierce ◽

José L. Lopez-Ribot ◽

Anand K. Ramasubramanian

Keyword(s):

Candida Albicans ◽

Antifungal Activity ◽

Biofilm Formation ◽

High Throughput Screening ◽

Fungal Infections ◽

Rapid Development ◽

Unmet Need ◽

Antifungal Drugs ◽

Content Type ◽

Pharmacologically Active

ABSTRACTCandida albicansis the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited and is particularly ineffective againstC. albicansbiofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors ofC. albicansbiofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics, or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents that inhibitC. albicansbiofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents, which are urgently needed.

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