Iron-sulfur protein odyssey: exploring their cluster functional versatility and challenging identification.

Iron-sulfur (Fe-S) clusters are an essential and ubiquitous class of protein-bound prosthetic centers that are involved in a broad range of biological processes (e.g. respiration, photosynthesis, DNA replication and repair and gene regulation) performing a wide range of functions including electron transfer, enzyme catalysis, and sensing. In a general manner, Fe-S clusters can gain or lose electrons through redox reactions, and are highly sensitive to oxidation, notably by small molecules such as oxygen and nitric oxide. The [2Fe-2S] and [4Fe-4S] clusters, the most common Fe-S cofactors, are typically coordinated by four amino acid side chains from the protein, usually cysteine thiolates, but other residues (e.g. histidine, aspartic acid) can also be found. While diversity in cluster coordination ensures the functional variety of the Fe-S clusters, the lack of conserved motifs makes new Fe-S protein identification challenging especially when the Fe-S cluster is also shared between two proteins as observed in several dimeric transcriptional regulators and in the mitoribosome. Thanks to the recent development of in cellulo, in vitro, and in silico approaches, new Fe-S proteins are still regularly identified, highlighting the functional diversity of this class of proteins. In this review, we will present three main functions of the Fe-S clusters and explain the difficulties encountered to identify Fe-S proteins and methods that have been employed to overcome these issues.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

Biochemical and cellular characterization of the CISD3 protein: Molecular bases of cluster release and destabilizing effects of nitric oxide.

The NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at -31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.

Cellular Responses and Targets in Food Spoilage Yeasts Exposed to Antifungal Prenylated Isoflavonoids.

Prenylated isoflavonoids are phytochemicals with promising antifungal properties. Recently, it was shown that glabridin and wighteone disrupted the plasma membrane (PM) of the food spoilage yeast Zygosaccharomyces parabailii in distinct ways, which led us to investigate further their modes of action (MoA). Transcriptomic profiling with Z. parabailii showed that genes encoding transmembrane ATPase transporters, including Yor1, and genes homologous to the pleiotropic drug resistance (PDR) subfamily in Saccharomyces cerevisiae were upregulated in response to both compounds. Gene functions involved in fatty acid and lipid metabolism, proteostasis, and DNA replication processes were overrepresented among genes upregulated by glabridin and/or wighteone. Chemogenomic analysis using the genome-wide deletant collection for S. cerevisiae further suggested an important role for PM lipids and PM proteins. Deletants of gene functions involved in biosynthesis of very-long-chain fatty acids (constituents of PM sphingolipids) and ergosterol were hypersensitive to both compounds. Using lipid biosynthesis inhibitors, we corroborated roles for sphingolipids and ergosterol in prenylated isoflavonoid action. The PM ABC transporter Yor1 and Lem3-dependent flippases conferred sensitivity and resistance, respectively, to the compounds, suggesting an important role for PM phospholipid asymmetry in their MoAs. Impaired tryptophan availability, likely linked to perturbation of the PM tryptophan permease Tat2, was evident in response to glabridin. Finally, substantial evidence highlighted a role of the endoplasmic reticulum (ER) in cellular responses to wighteone, including gene functions associated with ER membrane stress or with phospholipid biosynthesis, the primary lipid of the ER membrane. IMPORTANCE Preservatives, such as sorbic acid and benzoic acid, inhibit the growth of undesirable yeast and molds in foods. Unfortunately, preservative tolerance and resistance in food spoilage yeast, such as Zygosaccharomyces parabailii, is a growing challenge in the food industry, which can compromise food safety and increase food waste. Prenylated isoflavonoids are the main defense phytochemicals in the Fabaceae family. Glabridin and wighteone belong to this group of compounds and have shown potent antifungal activity against food spoilage yeasts. The present study demonstrated the mode of action of these compounds against food spoilage yeasts by using advanced molecular tools. Overall, the cellular actions of these two prenylated isoflavonoids share similarities (at the level of the plasma membrane) but also differences. Tryptophan import was specifically affected by glabridin, whereas endoplasmic reticulum membrane stress was specifically induced by wighteone. Understanding the mode of action of these novel antifungal agents is essential for their application in food preservation.

Redox-Based Strategies against Infections by Eukaryotic Pathogens.

Redox homeostasis is an equilibrium between reducing and oxidizing reactions within cells. It is an essential, dynamic process, which allows proper cellular reactions and regulates biological responses. Unbalanced redox homeostasis is the hallmark of many diseases, including cancer or inflammatory responses, and can eventually lead to cell death. Specifically, disrupting redox balance, essentially by increasing pro-oxidative molecules and favouring hyperoxidation, is a smart strategy to eliminate cells and has been used for cancer treatment, for example. Selectivity between cancer and normal cells thus appears crucial to avoid toxicity as much as possible. Redox-based approaches are also employed in the case of infectious diseases to tackle the pathogens specifically, with limited impacts on host cells. In this review, we focus on recent advances in redox-based strategies to fight eukaryotic pathogens, especially fungi and eukaryotic parasites. We report molecules recently described for causing or being associated with compromising redox homeostasis in pathogens and discuss therapeutic possibilities.

Novel Insights into Redox-Based Mechanisms for Auranofin-Induced Rapid Cancer Cell Death.

Auranofin (Ridaura®, AUF) is a gold complex originally approved as an antirheumatic agent that has emerged as a potential candidate for multiple repurposed therapies. The best-studied anticancer mechanism of AUF is the inhibition of thioredoxin reductase (TrxR). However, a number of reports indicate a more complex and multifaceted mode of action for AUF that could be cancer cell type- and dose-dependent. In this study, we observed that AUF displayed variable cytotoxicity in five triple-negative breast cancer cell lines. Using representative MDA-MB-231 cells treated with moderate and cytotoxic doses of AUF, we evidenced that an AUF-mediated TrxR inhibition alone may not be sufficient to induce cell death. Cytotoxic doses of AUF elicited rapid and drastic intracellular oxidative stress affecting the mitochondria, cytoplasm and nucleus. A "redoxome" proteomics investigation revealed that a short treatment with a cytotoxic dose AUF altered the redox state of a number of cysteines-containing proteins, pointing out that the cell proliferation/cell division/cell cycle and cell-cell adhesion/cytoskeleton structure were the mostly affected pathways. Experimentally, AUF treatment triggered a dose-dependent S-phase arrest and a rapid disintegration of the actin cytoskeleton structure. Our study shows a new spectrum of AUF-induced early effects and should provide novel insights into the complex redox-based mechanisms of this promising anticancer molecule.

Evolving challenges and strategies for fungal control in the food supply chain.

Fungi that spoil foods or infect crops can have major socioeconomic impacts, posing threats to food security. The strategies needed to manage these fungi are evolving, given the growing incidence of fungicide resistance, tightening regulations of chemicals use and market trends imposing new food-preservation challenges. For example, alternative methods for crop protection such as RNA-based fungicides, biocontrol, or stimulation of natural plant defences may lessen concerns like environmental toxicity of chemical fungicides. There is renewed focus on natural product preservatives and fungicides, which can bypass regulations for 'clean label' food products. These require investment to find effective, safe activities within complex mixtures such as plant extracts. Alternatively, physical measures may be one key for fungal control, such as polymer materials which passively resist attachment and colonization by fungi. Reducing or replacing traditional chlorine treatments (e.g. of post-harvest produce) is desirable to limit formation of disinfection by-products. In addition, the current growth in lower sugar food products can alter metabolic routing of carbon utilization in spoilage yeasts, with implications for efficacy of food preservatives acting via metabolism. The use of preservative or fungicide combinations, while involving more than one chemical, can reduce total chemicals usage where these act synergistically. Such approaches might also help target different subpopulations within heteroresistant fungal populations. These approaches are discussed in the context of current challenges for food preservation, focussing on pre-harvest fungal control, fresh produce and stored food preservation. Several strategies show growing potential for mitigating or reversing the risks posed by fungi in the food supply chain.

Inkjet 3D Printing of Polymers Resistant to Fungal Attachment.

Inkjet 3D printing is an additive manufacturing method that allows the user to produce a small batch of customized devices for comparative study versus commercial products. Here, we describe the use of a commercial 2D ink development system (Dimatix material printing) to manufacture small batches of 3D medical or other devices using a recently characterized fungal anti-attachment material. Such printed devices may resist problems that beset commercial medical products due to colonization by the fungal pathogen Candida albicans. By sequentially introducing the cross-section bitmaps of the product's CAD model and elevating the print head height using the auto-clicking script, we were able to create complex self-support geometries with the 2D ink development system. The use of this protocol allows researchers to produce a small batch of specimens for characterization from only a few grams of raw material. Additionally, we describe the testing of manufactured specimens for fungal anti-attachment. In comparison with most commercial AM systems, which require at least a few hundred grams of ink for printing trials, our protocol is well suited for smaller-scale production in material studies.

Potentiated inhibition of Trichoderma virens and other environmental fungi by new biocide combinations.

Fungi cause diverse, serious socio-economic problems, including biodeterioration of valuable products and materials that spawns a biocides industry worth ~$11 billion globally. To help combat environmental fungi that commonly colonise material products, this study tested the hypothesis that combination of an approved fungicide with diverse agents approved by the FDA (Food and Drug Administration) could reveal potent combinatorial activities with promise for fungicidal applications. The strategy to use approved compounds lowers potential development risks for any effective combinations. A high-throughput assay of 1280 FDA-approved compounds was conducted to find those that potentiate the effect of iodopropynyl-butyl-carbamate (IPBC) on the growth of Trichoderma virens; IPBC is one of the two most widely used Biocidal Products Regulations-approved fungicides. From this library, 34 compounds in combination with IPBC strongly inhibited fungal growth. Low-cost compounds that gave the most effective growth inhibition were tested against other environmental fungi that are standard biomarkers for resistance of synthetic materials to fungal colonisation. Trifluoperazine (TFZ) in combination with IPBC enhanced growth inhibition of three of the five test fungi. The antifungal hexetidine (HEX) potentiated IPBC action against two of the test organisms. Testable hypotheses on the mechanisms of these combinatorial actions are discussed. Neither IPBC + TFZ nor IPBC + HEX exhibited a combinatorial effect against mammalian cells. These combinations retained strong fungal growth inhibition properties after incorporation to a polymer matrix (alginate) with potential for fungicide delivery. The study reveals the potential of such approved compounds for novel combinatorial applications in the control of fungal environmental opportunists. KEY POINTS: • Search with an approved fungicide to find new fungicidal synergies in drug libraries. • New combinations inhibit growth of key environmental fungi on different matrices. • The approach enables a more rapid response to demand for new biocides.

Repurposing Nonantifungal Approved Drugs for Synergistic Targeting of Fungal Pathogens.

With the spread of drug resistance, new antimicrobials are urgently needed. Here, we set out to tackle this problem by high-throughput exploration for novel antifungal synergies among combinations of approved, nonantifungal drugs; a novel strategy exploiting the potential of alternative targets, low chemicals usage and low development risk. We screened the fungal pathogen Candida albicans by combining a small panel of nonantifungal drugs (all in current use for other clinical applications) with 1280 compounds from an approved drug library. Screens at sublethal concentrations of the antibiotic paromomycin (PM), the antimalarial primaquine (PQ), or the anti-inflammatory drug ibuprofen (IF) revealed a total of 17 potential strong, synergistic interactions with the library compounds. Susceptibility testing with the most promising combinations corroborated marked synergies [fractional inhibitory concentration (FIC) indices ≤0.5] between PM + ß-escin, PQ + celecoxib, and IF + pentamidine, reducing the MICs of PM, PQ, and IF in C. albicans by >64-, 16-, and 8-fold, respectively. Paromomycin + ß-escin and PQ + celecoxib were effective also against C. albicans biofilms, azole-resistant clinical isolates, and other fungal pathogens. Actions were specific, as no synergistic effect was observed in mammalian cells. Mode of action was investigated for one of the combinations, revealing that PM + ß-escin synergistically increase the error-rate of mRNA translation and suggesting a different molecular target to current antifungals. The study unveils the potential of the described combinatorial strategy in enabling acceleration of drug-repurposing discovery for combatting fungal pathogens.

Discovery of (meth)acrylate polymers that resist colonization by fungi associated with pathogenesis and biodeterioration.

Fungi have major, negative socioeconomic impacts, but control with bioactive agents is increasingly restricted, while resistance is growing. Here, we describe an alternative fungal control strategy via materials operating passively (i.e., no killing effect). We screened hundreds of (meth)acrylate polymers in high throughput, identifying several that reduce attachment of the human pathogen Candida albicans, the crop pathogen Botrytis cinerea, and other fungi. Specific polymer functional groups were associated with weak attachment. Low fungal colonization materials were not toxic, supporting their passive, anti-attachment utility. We developed a candidate monomer formulation for inkjet-based 3D printing. Printed voice prosthesis components showed up to 100% reduction in C. albicans biofilm versus commercial materials. Furthermore, spray-coated leaf surfaces resisted fungal infection, with no plant toxicity. This is the first high-throughput study of polymer chemistries resisting fungal attachment. These materials are ready for incorporation in products to counteract fungal deterioration of goods, food security, and health.

The Preservative Sorbic Acid Targets Respiration, Explaining the Resistance of Fermentative Spoilage Yeast Species.

A small number (10 to 20) of yeast species cause major spoilage in foods. Spoilage yeasts of soft drinks are resistant to preservatives like sorbic acid, and they are highly fermentative, generating large amounts of carbon dioxide gas. Conversely, many yeast species derive energy from respiration only, and most of these are sorbic acid sensitive and so prevented from causing spoilage. This led us to hypothesize that sorbic acid may specifically inhibit respiration. Tests with respirofermentative yeasts showed that sorbic acid was more inhibitory to both Saccharomyces cerevisiae and Zygosaccharomyces bailii during respiration (of glycerol) than during fermentation (of glucose). The respiration-only species Rhodotorula glutinis was equally sensitive when growing on either carbon source, suggesting that ability to ferment glucose specifically enables sorbic acid-resistant growth. Sorbic acid inhibited the respiration process more strongly than fermentation. We present a data set supporting a correlation between the level of fermentation and sorbic acid resistance across 191 yeast species. Other weak acids, C2 to C8, inhibited respiration in accordance with their partition coefficients, suggesting that effects on mitochondrial respiration were related to membrane localization rather than cytosolic acidification. Supporting this, we present evidence that sorbic acid causes production of reactive oxygen species, the formation of petite (mitochondrion-defective) cells, and Fe-S cluster defects. This work rationalizes why yeasts that can grow in sorbic acid-preserved foods tend to be fermentative in nature. This may inform more-targeted approaches for tackling these spoilage organisms, particularly as the industry migrates to lower-sugar drinks, which could favor respiration over fermentation in many spoilage yeasts.IMPORTANCE Spoilage by yeasts and molds is a major contributor to food and drink waste, which undermines food security. Weak acid preservatives like sorbic acid help to stop spoilage, but some yeasts, commonly associated with spoilage, are resistant to sorbic acid. Different yeasts generate energy for growth by the processes of respiration and/or fermentation. Here, we show that sorbic acid targets the process of respiration, so fermenting yeasts are more resistant. Fermentative yeasts are also those usually found in spoilage incidents. This insight helps to explain the spoilage of sorbic acid-preserved foods by yeasts and can inform new strategies for effective control. This is timely as the sugar content of products like soft drinks is being lowered, which may favor respiration over fermentation in key spoilage yeasts.

Mutational analysis of the Qi-site proton pathway in yeast cytochrome bc1 complex.

The respiratory cytochrome bc1 complex functions as a protonmotive ubiquinol:cytochrome c oxidoreductase. Lysine 228 (K228) located within the quinol reduction (Qi) site of the bc1 complex, has been reported as a key residue for proton transfer during the redox chemistry cycle to substrate quinone at Qi. In yeast, while single mutations had no effect, the combination of K228L and F225L resulted in a severe respiratory growth defect and inhibition of O2 consumption in intact cells. The inhibition was overcome by uncoupling the mitochondrial membrane or by suppressor mutations in the region of K228L-F225L. We propose that the K228L mutation introduces energetic (and kinetic) barriers into normal electron- and proton transfer chemistry at Qi, which are relieved by dissipation of the opposing protonmotive force or through the restoration of favourable intraprotein proton transfer networks via suppressor mutation.

Epoxy-amine oligomers from terpenes with applications in synergistic antifungal treatments.

A bis-epoxide monomer was synthesised in two steps from (R)-carvone, a terpenoid renewable feedstock derived from spearmint oil, and used to prepare ß-aminoalcohol oligomers in polyaddition reactions with bis-amines without requiring solvent or catalyst. A sub-set of the resultant materials were readily water soluble and were investigated for antifungal activity in combination with the fungicide iodopropynyl-butylcarbamate (IPBC) or the antifungal drug amphotericin B. The oligo-(ß-aminoalcohol)s alone were inactive against Trichoderma virens and Candida albicans but in combination with IPBC and amphotericin B demonstrated synergistic growth-inhibition of both fungi. Quantitative analysis showed that the presence of the terpene-based oligomers decreased the minimum inhibitory concentration (MIC) of IPBC by up to 64-fold and of amphotericin B by 8-fold. The efficacy of the combined formulation was further demonstrated with agar disk diffusion assays, which revealed that IPBC and amphotericin B reduced the growth of the fungi, as shown by zones of inhibition, to a greater extent when in the presence of the oligo-(ß-aminoalcohol)s. These data suggest potential future use of these renewable feedstock derived oligomers in antifungal material and related biomedical applications.

Novel Combinations of Agents Targeting Translation That Synergistically Inhibit Fungal Pathogens.

A range of fungicides or antifungals are currently deployed to control fungi in agriculture or medicine, but resistance to current agents is growing so new approaches and molecular targets are urgently needed. Recently, different aminoglycoside antibiotics combined with particular transport inhibitors were found to produce strong, synergistic growth-inhibition of fungi, by synergistically increasing the error rate of mRNA translation. Here, focusing on translation fidelity as a novel target for combinatorial antifungal treatment, we tested the hypothesis that alternative combinations of agents known to affect the availability of functional amino acids would synergistically inhibit growth of major fungal pathogens. We screened 172 novel combinations against three phytopathogens (Rhizoctonia solani, Zymoseptoria tritici, and Botrytis cinerea) and three human pathogens (Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus), showing that 48 combinations inhibited strongly the growth of the pathogens; the growth inhibition effect was significantly greater with the agents combined than by a simple product of their individual effects at the same doses. Of these, 23 combinations were effective against more than one pathogen, including combinations comprising food-and-drug approved compounds, e.g., quinine with bicarbonate, and quinine with hygromycin. These combinations [fractional inhibitory combination (FIC) index ≤0.5] gave up to 100% reduction of fungal growth yield at concentrations of agents which, individually, had negligible effect. No synergy was evident against bacterial, plant or mammalian cells, indicating specificity for fungi. Mode-of-action analyses for quinine + hygromycin indicated that synergistic mistranslation was the antifungal mechanism. That mechanism was not universal as bicarbonate exacerbated quinine action by increasing drug uptake. The study unveils chemical combinations and a target process with potential for control of diverse fungal pathogens, and suggests repurposing possibilities for several current therapeutics.

Heterologous Expression of a Novel Drug Transporter from the Malaria Parasite Alters Resistance to Quinoline Antimalarials.

Antimalarial drug resistance hampers effective malaria treatment. Critical SNPs in a particular, putative amino acid transporter were recently linked to chloroquine (CQ) resistance in malaria parasites. Here, we show that this conserved protein (PF3D7_0629500 in Plasmodium falciparum; AAT1 in P. chabaudi) is a structural homologue of the yeast amino acid transporter Tat2p, which is known to mediate quinine uptake and toxicity. Heterologous expression of PF3D7_0629500 in yeast produced CQ hypersensitivity, coincident with increased CQ uptake. PF3D7_0629500-expressing cultures were also sensitized to related antimalarials; amodiaquine, mefloquine and particularly quinine. Drug sensitivity was reversed by introducing a SNP linked to CQ resistance in the parasite. Like Tat2p, PF3D7_0629500-dependent quinine hypersensitivity was suppressible with tryptophan, consistent with a common transport mechanism. A four-fold increase in quinine uptake by PF3D7_0629500 expressing cells was abolished by the resistance SNP. The parasite protein localised primarily to the yeast plasma membrane. Its expression varied between cells and this heterogeneity was used to show that high-expressing cell subpopulations were the most drug sensitive. The results reveal that the PF3D7_0629500 protein can determine the level of sensitivity to several major quinine-related antimalarials through an amino acid-inhibitable drug transport function. The potential clinical relevance is discussed.

Mitochondrial Ferredoxin Determines Vulnerability of Cells to Copper Excess.

The essential micronutrient copper is tightly regulated in organisms, as environmental exposure or homeostasis defects can cause toxicity and neurodegenerative disease. The principal target(s) of copper toxicity have not been pinpointed, but one key effect is impaired supply of iron-sulfur (FeS) clusters to the essential protein Rli1 (ABCE1). Here, to find upstream FeS biosynthesis/delivery protein(s) responsible for this, we compared copper sensitivity of yeast-overexpressing candidate targets. Overexpression of the mitochondrial ferredoxin Yah1 produced copper hyper-resistance. 55Fe turnover assays revealed that FeS integrity of Yah1 was particularly vulnerable to copper among the test proteins. Furthermore, destabilization of the FeS domain of Yah1 produced copper hypersensitivity, and YAH1 overexpression rescued Rli1 dysfunction. This copper-resistance function was conserved in the human ferredoxin, Fdx2. The data indicate that the essential mitochondrial ferredoxin is an important copper target, determining a tipping point where plentiful copper supply becomes excessive. This knowledge could help in tackling copper-related diseases.

The Candidate Antimalarial Drug MMV665909 Causes Oxygen-Dependent mRNA Mistranslation and Synergizes with Quinoline-Derived Antimalarials.

To cope with growing resistance to current antimalarials, new drugs with novel modes of action are urgently needed. Molecules targeting protein synthesis appear to be promising candidates. We identified a compound (MMV665909) from the Medicines for Malaria Venture (MMV) Malaria Box of candidate antimalarials that could produce synergistic growth inhibition with the aminoglycoside antibiotic paromomycin, suggesting a possible action of the compound in mRNA mistranslation. This mechanism of action was substantiated with a Saccharomyces cerevisiae model using available reporters of mistranslation and other genetic tools. Mistranslation induced by MMV665909 was oxygen dependent, suggesting a role for reactive oxygen species (ROS). Overexpression of Rli1 (a ROS-sensitive, conserved FeS protein essential in mRNA translation) rescued inhibition by MMV665909, consistent with the drug's action on translation fidelity being mediated through Rli1. The MMV drug also synergized with major quinoline-derived antimalarials which can perturb amino acid availability or promote ROS stress: chloroquine, amodiaquine, and primaquine. The data collectively suggest translation fidelity as a novel target of antimalarial action and support MMV665909 as a promising drug candidate.

Metal-Based Combinations That Target Protein Synthesis by Fungi.

A wide range of fungicides (or antifungals) are used in agriculture and medicine, with activities against a spectrum of fungal pathogens. Unfortunately, the evolution of fungicide resistance has become a major issue. Therefore, there is an urgent need for new antifungal treatments. Certain metals have been used for decades as efficient fungicides in agriculture. However, concerns over metal toxicity have escalated over this time. Recent studies have revealed that metals like copper and chromate can impair functions required for the fidelity of protein synthesis in fungi. This occurs through different mechanisms, based on targeting of iron-sulphur cluster integrity or competition for uptake with amino acid precursors. Moreover, chromate at least acts synergistically with other agents known to target translation fidelity, like aminoglycoside antibiotics, causing dramatic and selective growth inhibition of several fungal pathogens of humans and plants. As such synergy allows the application of decreased amounts of metals for effective inhibition, it lessens concerns about nonspecific toxicity and opens new possibilities for metal applications in combinatorial fungicides targeting protein synthesis.

Human Mitochondrial Cytochrome b Variants Studied in Yeast: Not All Are Silent Polymorphisms.

Variations in mitochondrial DNA (mtDNA) cytochrome b (mt-cyb) are frequently found within the healthy population, but also occur within a spectrum of mitochondrial and common diseases. mt-cyb encodes the core subunit (MT-CYB) of complex III, a central component of the oxidative phosphorylation system that drives cellular energy production and homeostasis. Despite significant efforts, most mt-cyb variations identified are not matched with corresponding biochemical data, so their functional and pathogenic consequences in humans remain elusive. While human mtDNA is recalcitrant to genetic manipulation, it is possible to introduce human-associated point mutations into yeast mtDNA. Using this system, we reveal direct links between human mt-cyb variations in key catalytic domains of MT-CYB and significant changes to complex III activity or drug sensitivity. Strikingly, m.15257G>A (p.Asp171Asn) increased the sensitivity of yeast to the antimalarial drug atovaquone, and m.14798T>C (p.Phe18Leu) enhanced the sensitivity of yeast to the antidepressant drug clomipramine. We demonstrate that while a small number of mt-cyb variations had no functional effect, others have the capacity to alter complex III properties, suggesting they could play a wider role in human health and disease than previously thought. This compendium of new mt-cyb-biochemical relationships in yeast provides a resource for future investigations in humans.