Potential Antifungal Targets Research Articles

Structural insights into the antifungal drug target guanosine monophosphate synthase from Aspergillus fumigatus.

Purine biosynthesis is a fundamental cellular process that sustains life by maintaining the intracellular pool of purines for DNA/RNA synthesis and signal transduction. As an integral determinant of fungal survival and virulence, the enzymes in this metabolic pathway have been pursued as potential antifungal targets. Guanosine monophosphate (GMP) synthase has been identified as an attractive target as it is essential for virulence in the clinically prominent fungal pathogens Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. However, a lack of structural information on GMP synthase has hindered drug-design efforts. Here, the first structure of a GMP synthase of fungal origin, that from A. fumigatus (at 2.3 Å resolution), is presented. Structural analysis of GMP synthase shows a distinct absence of the D1 dimerization domain that is present in the human homologue. Interestingly, A. fumigatus GMP synthase adopts a dimeric state, as determined by native mass spectrometry and gel-filtration chromatography, in contrast to the monomeric human homologue. Analysis of the substrate-binding pockets of A. fumigatus GMP synthase reveals key differences in the ATP- and XMP-binding sites that can be exploited for species-specific inhibitor drug design. Furthermore, the inhibitory activities of the glutamine analogues acivicin (IC50 = 16.6 ± 2.4 µM) and 6-diazo-5-oxo-L-norleucine (IC50 = 29.6 ± 5.6 µM) against A. fumigatus GMP synthase are demonstrated. Together, these data provide crucial structural information required for specifically targeting A.fumigatus GMP synthase for future antifungal drug-discovery endeavours.

Leveraging machine learning essentiality predictions and chemogenomic interactions to identify antifungal targets

Fungal pathogens pose a global threat to human health, with Candida albicans among the leading killers. Systematic analysis of essential genes provides a powerful strategy to discover potential antifungal targets. Here, we build a machine learning model to generate genome-wide gene essentiality predictions for C. albicans and expand the largest functional genomics resource in this pathogen (the GRACE collection) by 866 genes. Using this model and chemogenomic analyses, we define the function of three uncharacterized essential genes with roles in kinetochore function, mitochondrial integrity, and translation, and identify the glutaminyl-tRNA synthetase Gln4 as the target of N-pyrimidinyl-β-thiophenylacrylamide (NP-BTA), an antifungal compound.

Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads

Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and amphotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have Ki values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.

Opportunistic yeast pathogenCandidaspp.: Secreted and membrane-bound virulence factors

Candida virulence factors (VFs) including mainly enzymes and proteins play vital roles in breaching the human intestinal barrier and causing deadly invasive candidiasis. Limited VFs' structural studies hinder deeper comprehension of their mechanisms and thus the design of vaccines and antifungal drugs against fungal infections.

The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure.

The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.

Functional dissection of individual domains in group III histidine kinase Sshk1p from the phytopathogenic fungus Sclerotinia sclerotiorum

A conserved kinase domain and phosphoryl group receiver domain at the C-terminus and poly-HAMP domains at the N-terminus comprise the structural components of the group III HK which was considered as a potential antifungal target. However, the roles of individual domains in the function of group III HKs have rarely been dissected in fungi. In this study, we dissected the roles of individual domains to better understand the function of Sshk1p, a group III HK from Sclerotinia sclerotiorum. The results suggest that individual domains play different roles in the functionality of Sshk1p and are implicated in the regulation of mycelial growth, sclerotia formation, pathogenicity. And the mutants of each domain in Sshk1 showed significantly increased sensitivity to hyperosmotic stress. However, the mutants of each domain in Sshk1 showed high resistance to fludioxonil and dimethachlon which suggested that all nine domains of Sshk1p were indispensable for susceptibility to fludioxonil and dimethachlon. Moreover, deletion of each individual domain in Sshk1 cancelled intracellular glycerol accumulation and increased SsHog1p phosphorylation level triggered by NaCl and fludioxonil, suggesting that all the domains of Sshk1 were essential for Sshk1-mediated SsHog1p phosphorylation and subsequent polyol accumulation in response to fludioxonil and hyperosmotic stress.

Duplication and Functional Divergence of Branched-Chain Amino Acid Biosynthesis Genes in Aspergillus nidulans.

ABSTRACTFungi, bacteria, and plants, but not animals, synthesize the branched-chain amino acids: leucine, isoleucine, and valine. While branched-chain amino acid (BCAA) biosynthesis has been well characterized in the yeast Saccharomyces cerevisiae, it is incompletely understood in filamentous fungi. The three BCAAs share several early biosynthesis steps before divergence into specific pathways. In Aspergillus nidulans, the genes for the first two dedicated steps in leucine biosynthesis have been characterized, but the final two have not. We used sequence searches of the A. nidulans genome to identify two genes encoding β-isopropylmalate dehydrogenase, which catalyzes the penultimate step of leucine biosynthesis, and six genes encoding BCAA aminotransferase, which catalyzes the final step in biosynthesis of all three BCAA. We have used combinations of gene knockouts to determine the relative contribution of each of these genes to BCAA biosynthesis. While both β-isopropylmalate dehydrogenase genes act in leucine biosynthesis, the two most highly expressed BCAA aminotransferases are responsible for BCAA biosynthesis. We have also characterized the expression of leucine biosynthesis genes using reverse transcriptase-quantitative PCR and found regulation in response to leucine availability is mediated through the Zn(II)2Cys6 transcription factor LeuB.

Fungal homoserine transacetylase: A potential antifungal target

We are facing a significant challenge due to the tremendous rise in drug resistant fungal pathogen populations. The number of antifungal drugs is limited, as are the known targets they inhibit. The limited number of drugs and targets that have been successfully exploited in antifungal drug discovery is in part due to the eukaryotic nature of these organisms, which is shared with mammalian cells. An ideal antifungal substance is one that targets an indispensable pathway for pathogenic fungal survival that is not present in mammals or plants. Methionine is an essential amino acid for fungi and inhibition of its biosynthesis is deleterious for the pathogen. The biochemical pathways that produce methionine do not exist in mammals as they acquire it from their diet. Homoserine transacetylase, which catalyses the first committed step in methionine biosynthesis, therefore represents an attractive drug target. This enzyme is absent in mammals and unique to fungi and bacteria, suggesting that drugs targeting it would be of low toxicity, if any, to mammals, while having a lethal effect against the fungal pathogen. Homoserine transacetylase, based on its role in the production of methionine and its absence in mammals and plants could be a potential antifungal target.

Identifying Conserved Generic Aspergillus spp. Co-Expressed Gene Modules Associated with Germination Using Cross-Platform and Cross-Species Transcriptomics.

Aspergillus spp. is an opportunistic human pathogen that may cause a spectrum of pulmonary diseases. In order to establish infection, inhaled conidia must germinate, whereby they break dormancy, start to swell, and initiate a highly polarized growth process. To identify critical biological processes during germination, we performed a cross-platform, cross-species comparative analysis of germinating A. fumigatus and A. niger conidia using transcriptional data from published RNA-Seq and Affymetrix studies. A consensus co-expression network analysis identified four gene modules associated with stages of germination. These modules showed numerous shared biological processes between A. niger and A. fumigatus during conidial germination. Specifically, the turquoise module was enriched with secondary metabolism, the black module was highly enriched with protein synthesis, the darkgreen module was enriched with protein fate, and the blue module was highly enriched with polarized growth. More specifically, enriched functional categories identified in the blue module were vesicle formation, vesicular transport, tubulin dependent transport, actin-dependent transport, exocytosis, and endocytosis. Genes important for these biological processes showed similar expression patterns in A. fumigatus and A. niger, therefore, they could be potential antifungal targets. Through cross-platform, cross-species comparative analysis, we were able to identify biologically meaningful modules shared by A. fumigatus and A. niger, which underscores the potential of this approach.

Genetic and structural validation of phosphomannomutase as a cell wall target in Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic mold responsible for severe life-threatening fungal infections in immunocompromised patients. The cell wall, an essential structure composed of glucan, chitin, and galactomannan, is considered to be a target for the development of antifungal drugs. The nucleotide sugar donor GDP-mannose (GDP-Man) is required for the biosynthesis of galactomannan, glycosylphosphatidylinositol (GPI) anchors, glycolipid, and protein glycosylation. Starting from fructose-6-phosphate, GDP-Man is produced by the sequential action of the enzymes phosphomannose isomerase, phosphomannomutase (Pmm), and GDP-mannose pyrophosphorylase. Here, using heterokaryon rescue and gene knockdown approaches we demonstrate that the phosphomannomutase encoding gene in A. fumigatus (pmmA) is essential for survival. Reduced expression of pmmA is associated with significant morphological defects including retarded germination, growth, reduced conidiation, and abnormal polarity. Moreover, the knockdown strain exhibited an altered cell wall organization and sensitivity toward cell wall perturbing agents. By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non-conservative substitutions near the active site when compared to the human orthologues. Taken together, this work provides a genetic and structural foundation for the exploitation of AfPmmA as a potential antifungal target.

Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: essential tRNA splicing enzymes and potential anti-fungal targets.

The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2′-PO4 generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2′-PO4 attacks NAD+ to form an RNA-2′-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Because Tpt1 does not participate in metazoan tRNA splicing, and Tpt1 knockout has no apparent impact on mammalian physiology, Tpt1 is considered a potential antifungal drug target. Here we characterize Tpt1 enzymes from four human fungal pathogens: Coccidioides immitis, the agent of Valley Fever; Aspergillus fumigatus and Candida albicans, which cause invasive, often fatal, infections in immunocompromised hosts; and Candida auris, an emerging pathogen that is resistant to current therapies. All four pathogen Tpt1s were active in vivo in complementing a lethal Saccharomyces cerevisiae tpt1Δ mutation and in vitro in NAD+-dependent conversion of a 2′-PO4 to a 2′-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu of NAD+, albeit with much lower affinity, whereas nicotinic acid adenine dinucleotide was ineffective. Fungal Tpt1s efficiently removed an internal ribonucleotide 2′-phosphate from an otherwise all-DNA substrate. Replacement of an RNA ribose-2′-PO4 nucleotide with arabinose-2′-PO4 diminished enzyme specific activity by ≥2000-fold and selectively slowed step 2 of the reaction pathway, resulting in transient accumulation of an ara-2′-phospho-ADP-ribosylated intermediate. Our results implicate the 2′-PO4 ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.

Targeting Unconventional Pathways in Pursuit of Novel Antifungals

The impact of invasive fungal infections on human health is a serious, but largely overlooked, public health issue. Commonly affecting the immunocompromised community, fungal infections are predominantly caused by species of Candida, Cryptococcus, and Aspergillus. Treatments are reliant on the aggressive use of pre-existing antifungal drug classes that target the fungal cell wall and membrane. Despite their frequent use, these drugs are subject to unfavorable drug-drug interactions, can cause undesirable side-effects and have compromised efficacy due to the emergence of antifungal resistance. Hence, there is a clear need to develop novel classes of antifungal drugs. A promising approach involves exploiting the metabolic needs of fungi by targeted interruption of essential metabolic pathways. This review highlights potential antifungal targets including enolase, a component of the enolase-plasminogen complex, and enzymes from the mannitol biosynthesis and purine nucleotide biosynthesis pathways. There has been increased interest in the enzymes that comprise these particular pathways and further investigation into their merits as antifungal targets and roles in fungal survival and virulence are warranted. Disruption of these vital processes by targeting unconventional pathways with small molecules or antibodies may serve as a promising approach to discovering novel classes of antifungals.

Identification and characterization of a sulfite reductase gene and new insights regarding the sulfur-containing amino acid metabolism in the basidiomycetous yeast Cryptococcus neoformans.

The amino acid biosynthetic pathway of invasive pathogenic fungi has been studied as a potential antifungal drug target. Studies of the disruption of genes involved in amino acid biosynthesis have demonstrated the importance of this pathway in the virulence of Cryptococcus neoformans. Here, we identified the MET5 (CNL05500) and MET10 (CNG03990) genes in this pathway, both encoding sulfite reductase, which catalyzes the reduction of sulfite to sulfide. The MET14 (CNE03880) gene was also identified, which is responsible for the conversion of sulfate to sulfite. The use of cysteine as a sulfur source led to the production of methionine via hydrogen sulfide synthesis mediated by CYS4 (CNA06170), CYS3 (CNN01730), and MST1 (CND03690). MST1 exhibited high homology with the TUM1 gene of Saccharomyces cerevisiae, which has functional similarity with the 3-mercaptopyruvate sulfurtransferase (3-MST) gene in humans. Although the hypothesis that hydrogen sulfide is produced from cysteine via CYS4, CYS3, and MST1 warrants further study, the new insight into the metabolic pathway of sulfur-containing amino acids in C. neoformans provided here indicates the usefulness of this system in the development of screening tools for antifungal drug agents.

Histone acetylation/deacetylation in Candida albicans and their potential as antifungal targets.

Recently, the incidence of invasive fungal infections has significantly increased. Candida albicans (C. albicans) is the most common opportunistic fungal pathogen that infects humans. The limited number of available antifungal agents and the emergence of drug resistance pose difficulties to treatment, thus new antifungals are urgently needed. Through their functions in DNA replication, DNA repair and transcription, histone acetyltransferases (HATs) and histone deacetylases (HDACs) perform essential functions relating to growth, virulence, drug resistance and stress responses of C. albicans. Here, we summarize the physiological and pathological functions of HATs/HDACs, potential antifungal targets and underlying antifungal compounds that impact histone acetylation and deacetylation. We anticipate this review will stimulate the identification of new HAT/HDAC-related antifungal targets and antifungal agents.

Potential Antifungal Targets Based on Glucose Metabolism Pathways of Candida albicans.

In recent years, fungal infections have become a serious health problem. Candida albicans are considered as the fourth most common isolates associated with approximately 40% mortality in bloodstream infections among hospitalized patients. Due to various limitations of classical antifungals used currently, such as limited kinds of drugs, inevitable toxicities, and high price, there is an urgent need to explore new antifungal agents based on novel targets. Generally, nutrient metabolism is involved with fungal virulence, and glucose is one of the important nutrients in C. albicans. C. albicans can obtain and metabolize glucose through a variety of pathways; in theory, many enzymes in these pathways can be potential targets for developing new antifungal agents, and several studies have confirmed that compounds which interfere with alpha-glucosidase, acid trehalase, trehalose-6-phosphate synthase, class II fructose bisphosphate aldolases, and glucosamine-6-phosphate synthase in these pathways do have antifungal activities. In this review, the glucose metabolism pathways in C. albicans, the potential antifungal targets based on these pathways, and some compounds which have antifungal activities by inhibiting several enzymes in these pathways are summarized. We believe that our review will be helpful to the exploration of new antifungal drugs with novel antifungal targets.

PP2A-Like Protein Phosphatase (Sit4) Regulatory Subunits, Sap155 and Sap190, Regulate Candida albicans' Cell Growth, Morphogenesis, and Virulence.

PP2A-like phosphatases share high homology with PP2A enzymes and are composed of a catalytic subunit and a regulatory subunit. In Candida albicans, the PP2A-like catalytic subunit SIT4 regulates cell growth, morphogenesis, and virulence. However, the functions of its regulatory subunits remain unclear. Here, by homology analysis and co-IP experiments, we identified two regulatory subunits of SIT4 in C. albicans, SAP155 (orf19.642) and SAP190 (orf19.5160). We constructed sit4Δ/Δ, sap155Δ/Δ, sap190Δ/Δ, and sap155Δ/Δ sap190Δ/Δ mutants and found that deleting SAP155 had no apparent phenotypic consequence, while deleting SAP190 caused slow growth, hypersensitivity to cell wall stress, abnormal morphogenesis in response to serum or genotoxic stress (HU and MMS), less damage to macrophages, and attenuated virulence in mice. However, deleting both SAP155 and SAP190 caused significantly stronger defects, which was similar to deleting SIT4. Together, our results suggest that SAP190 is required for the function of SIT4 and that SAP155 can partially compensate for the loss of SAP190 in C. albicans. Given the vital role of these regulatory subunits of SIT4 in C. albicans physiology and virulence, they could serve as potential antifungal targets.

Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.

A Histone Acetyltransferase Inhibitor with Antifungal Activity against CTG clade Candida Species.

Candida species represent one of the most frequent causes of hospital-acquired infections in immunocompromised patient cohorts. Due to a very limited set of antifungals available and an increasing prevalence of drug resistance, the discovery of novel antifungal targets is essential. Targeting chromatin modifiers as potential antifungal targets has gained attention recently, mainly due to their role in regulating virulence in Candida species. Here, we describe a novel activity for the histone acetyltransferase inhibitor Cyclopentylidene-[4-(4-chlorophenyl)thiazol-2-yl)hydrazone (CPTH2) as a specific inhibitor of CTG clade Candida species. Furthermore, we show that CPTH2 has fungicidal activity and protects macrophages from Candida-mediated death. Thus, this work could provide a starting point for the development of novel antifungals specific to CTG clade Candida species.

The PP2A regulatory subunits, Cdc55 and Rts1, play distinct roles in Candida albicans’ growth, morphogenesis, and virulence

Protein phosphatase-2A (PP2A) is a heterotrimeric enzyme composed of a catalytic subunit, a regulatory subunit, and a structural subunit. In Candida albicans, Cdc55 and Rts1 have been identified as possible regulatory subunits of PP2A containing the catalytic subunit Pph21 and structural subunit Tpd3. The Tpd3-Pph21 phosphatase regulates cell morphogenesis and division. However, the functions of Cdc55 and Rts1 remain unclear. Here, we constructed cdc55Δ/Δ and rts1Δ/Δ mutants and found that they exhibit different defects in multiple phenotypes although both show similar hyperphosphorylation of the septin Sep7 and aberrant septin organization. Under yeast growth conditions, the cdc55Δ/Δ mutant grows slowly as pseudohyphae with some cells lacking the nucleus, while rts1Δ/Δ cells are round and enlarged and seem to undergo incomplete cell separation producing multinucleated cells. Strong chitin deposition occurs at the septum of cdc55Δ/Δ cells and on the surface of rts1Δ/Δ cells, which likely contributes to increased susceptibility to caspofungin. Also, cdc55Δ/Δ exhibits severe defects in hyphal and biofilm formation, while rts1Δ/Δ is partially defective. Both mutants show reduced virulence in mice, suggesting that PP2A-B subunits could serve as potential antifungal targets.

Characterization of the atypical Ppz/Hal3 phosphatase system from the pathogenic fungus Cryptococcus neoformans.

Ppz Ser/Thr protein phosphatases (PPases) are found only in fungi and have been proposed as potential antifungal targets. In Saccharomyces cerevisiae Ppz1 (ScPpz1) is involved in regulation of monovalent cation homeostasis. ScPpz1 is inhibited by two regulatory proteins, Hal3 and Vhs3, which have moonlighting properties, contributing to the formation of an unusual heterotrimeric PPC decarboxylase (PPCDC) complex crucial for CoA biosynthesis. Here we report the functional characterization of CnPpz1 (CNAG_03673) and two possible Hal3-like proteins, CnHal3a (CNAG_00909) and CnHal3b (CNAG_07348) from the pathogenic fungus Cryptococcus neoformans. Deletion of CnPpz1 or CnHal3b led to phenotypes unrelated to those observed in the equivalent S. cerevisiae mutants, and the CnHal3b-deficient strain was less virulent. CnPpz1 is a functional PPase and partially replaced endogenous ScPpz1. Both CnHal3a and CnHal3b interact with ScPpz1 and CnPpz1 in vitro but do not inhibit their phosphatase activity. Consistently, when expressed in S. cerevisiae, they poorly reproduced the Ppz1-regulatory properties of ScHal3. In contrast, both proteins were functional monogenic PPCDCs. The CnHal3b isoform was crystallized and, for the first time, the 3D-structure of a fungal PPCDC elucidated. Therefore, our work provides the foundations for understanding the regulation and functional role of the Ppz1-Hal3 system in this important pathogenic fungus.