Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Synthesis and Evaluation of Structurally Diverse C-2-Substituted Thienopyrimidine-Based Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase.

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.

Inhibitory Potential of New Phenolic Hydrazide-Hydrazones with a Decoy Substrate Fragment towards Laccase from a Phytopathogenic Fungus: SAR and Molecular Docking Studies.

Laccase from pathogenic fungi participates in both the delignification and neutralization of phytoantibiotics. Furthermore, it interferes with the hormone signaling in plants and catalyzes melanization. Infections of these pathogens contribute to loss in forestry, agriculture, and horticulture. As there is still a need to expand knowledge on efficient defense strategies against phytopathogenic fungi, the present study aimed to reveal more information on the molecular mechanisms of laccase inhibition with natural and natural-like carboxylic acid semi-synthetic derivatives. A set of hydrazide-hydrazones derived from carboxylic acids, generally including electron-rich arene units that serve as a decoy substrate, was synthesized and tested with laccase from Trametes versicolor. The classic synthesis of the title inhibitors proceeded with good to almost quantitative yield. Ninety percent of the tested molecules were active in the range of KI = 8-233 µM and showed different types of action. Such magnitude of inhibition constants qualified the hydrazide-hydrazones as strong laccase inhibitors. Molecular docking studies supporting the experimental data explained the selected derivatives' interactions with the enzyme. The results are promising in developing new potential antifungal agents mitigating the damage scale in the plant cultivation, gardening, and horticulture sectors.

The Short-Term Exposure to SDHI Fungicides Boscalid and Bixafen Induces a Mitochondrial Dysfunction in Selective Human Cell Lines.

Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.

Wells-Dawson phosphotungstates as mushroom tyrosinase inhibitors: a speciation study.

In order to elucidate the active polyoxotungstate (POT) species that inhibit fungal polyphenol oxidase (AbPPO4) in sodium citrate buffer at pH 6.8, four Wells-Dawson phosphotungstates [α/ß-PV2WVI18O62]6- (intact form), [α2-PV2WVI17O61]10- (monolacunary), [PV2WVI15O56]12- (trilacunary) and [H2PV2WVI12O48]12- (hexalacunary) were investigated. The speciation of the POT solutions under the dopachrome assay (50 mM Na-citrate buffer, pH 6.8; L-3,4-dihydroxyphenylalanine as a substrate) conditions were determined by 183W-NMR, 31P-NMR spectroscopy and mass spectrometry. The intact Wells-Dawson POT [α/ß-PV2WVI18O62]6- shows partial (~ 69%) disintegration into the monolacunary [α2-PV2WVI17O61]10- anion with moderate activity (Ki = 9.7 mM). The monolacunary [α2-PV2WVI17O61]10- retains its structural integrity and exhibits the strongest inhibition of AbPPO4 (Ki = 6.5 mM). The trilacunary POT [PV2WVI15O56]12- rearranges to the more stable monolacunary [α2-PV2WVI17O61]10- (~ 62%) accompanied by release of free phosphates and shows the weakest inhibition (Ki = 13.6 mM). The hexalacunary anion [H2PV2WVI12O48]12- undergoes time-dependent hydrolysis resulting in a mixture of [H2PV2WVI12O48]12-, [PV8WVI48O184]40-, [PV2WVI19O69(H2O)]14- and [α2-PV2WVI17O61]10- which together leads to comparable inhibitory activity (Ki = 7.5 mM) after 48 h. For the solutions of [α/ß-PV2WVI18O62]6-, [α2-PV2WVI17O61]10- and [PV2WVI15O56]12- the inhibitory activity is correlated to the degree of their rearrangement to [α2-PV2WVI17O61]10-. The rearrangement of hexalacunary [H2PV2WVI12O48]12- into at least four POTs with a negligible amount of monolacunary anion interferes with the correlation of activity to the degree of their rearrangement to [α2-PV2WVI17O61]10-. The good inhibitory effect of the Wells-Dawson [α2-PV2WVI17O61]10- anion is explained by the low charge density of its protonated forms Hx[α2-PV2WVI17O61](10-x)- (x = 3 or 4) at pH 6.8.

Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

Prediction of hyaluronic acid target on sucrase-isomaltase (SI) with reverse docking and molecular dynamics simulations for inhibitors binding to SI.

Auricularia cornea (E.) polysaccharide is an important component of A. cornea Ehrenb, a white mutant strain of Auricularia with biological activities, such as enhancement of human immune function and cancer prevention. The hyaluronic acids (HAs) are important components of the A. cornea polysaccharide and have extremely high medicinal value. In this study, we used HA to search the target protein sucrase-isomaltase (SI). In addition, we also performed molecular dynamics (MD) simulations to explore the binding of three inhibitors (HA, acarbose and kotalanol) to SI. The MD simulations indicated that the binding of the three inhibitors may induce the partial disappearance of α helix in residues 530-580. Hence, the hydrogen bond for Gly570-Asn572, which was near the catalytic base Asp471 in SI, was broken during the binding of the three inhibitors. We reveal a new inhibitor for SI and provide reasonable theoretical clues for inhibitor binding to SI.

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety.

We previously reported (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the ß-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,ß-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC50 = 18.09 ± 0.25 µM), 1h (IC50 = 4.14 ± 0.10 µM), and 2a (IC50 = 15.69 ± 0.40 µM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC50 = 48.62 ± 3.38 µM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.

Structure-Guided Design of the First Noncovalent Small-Molecule Inhibitor of CRM1.

Nuclear export factor chromosome region maintenance 1 (CRM1) is an attractive anticancer and antiviral drug target that spurred several research efforts to develop its inhibitor. Noncovalent CRM1 inhibitors are desirable, but none is reported to date. Here, we present the crystal structure of yeast CRM1 in complex with S109, a substructure of CBS9106 (under clinical test). Superimposition with the LFS-829 (another covalent CRM1 inhibitor) complex inspired the design of a noncovalent CRM1 inhibitor. Among nine synthesized compounds, noncovalent CRM1 inhibitor 1 (NCI-1) showed a high affinity to human and yeast CRM1 in the absence or presence of GST-bound Ras-related nuclear protein (RanGTP). Unlike covalent inhibitors, the crystal structure showed that NCI-1 is bound in the "open" nuclear export signal (NES) groove of CRM1, simultaneously occupying two hydrophobic pockets. NCI-1 additionally inhibited the nuclear export and proliferation of cells harboring the human CRM1-C528S mutant. Our work opens up the avenue of noncovalent CRM1 inhibitor development toward a more potent, less toxic, and broad-spectrum anticancer/antiviral therapy.

Re-emerging Aspartic Protease Targets: Examining Cryptococcus neoformans Major Aspartyl Peptidase 1 as a Target for Antifungal Drug Discovery.

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

Deletion of the natural inhibitory protein Inh1 in Ustilago maydis has no effect on the dimeric state of the F1FO-ATP synthase but increases the ATPase activity and reduces the stability.

Transduction of electrochemical proton gradient into ATP synthesis is performed by F1FO-ATP synthase. The reverse reaction is prevented by the regulatory subunit Inh1. Knockout of the inh1 gene in the basidiomycete Ustilago maydis was generated in order to study the function of this protein in the mitochondrial metabolism and cristae architecture. Deletion of inh1 gen did not affect cell growth, glucose consumption, and biomass production. Ultrastructure and fluorescence analyzes showed that size, cristae shape, network, and distribution of mitochondria was similar to wild strain. Membrane potential, ATP synthesis, and oxygen consumption in wild type and mutant strains had similar values. Kinetic analysis of ATPase activity of complex V in permeabilized mitochondria showed similar values of Vmax and KM for both strains, and no effect of pH was observed. Interestingly, the dimeric state of complex V occurs in the mutant strain, indicating that this subunit is not essential for dimerization. ATPase activity of the isolated monomeric and dimeric forms of complex V indicated Vmax values 4-times higher for the mutant strain than for the WT strain, suggesting that the absence of Inh1 subunit increased ATPase activity, and supporting a regulatory role for this protein; however, no effect of pH was observed. ATPase activity of WT oligomers was stimulated several times by dodecyl-maltoside (DDM), probably by removal of ADP from F1 sector, while DDM induced an inactive form of the mutant oligomers.

Health Potential of Clery Strawberries: Enzymatic Inhibition and Anti-Candida Activity Evaluation.

Strawberries, belonging to cultivar Clery (Fragaria × ananassa Duchesne ex Weston) and to a graft obtained by crossing Clery and Fragaria vesca L., were chosen for a study on their health potential, with regard to the prevention of chronic and degenerative diseases. Selected samples, coming from fresh and defrosted berries, submitted to different homogenization techniques combined with thermal and microwave treatments, had been previously analyzed in their polyphenolic content and antioxidant capacity. In the present work, these homogenates were evaluated in relation to their enzymatic inhibition activity towards acetylcholinesterase and butyrylcholinesterase, α-amylase, α-glucosidase and tyrosinase. All these enzymes, involved in the onset of diabetes, and neurodegenerative and other chronic diseases, were modulated by the tested samples. The inhibitory effect on tyrosinase and cholinesterase was the most valuable. Antifungal activity against Candida albicans, recently shown to play a crucial role in human gut diseases as well as diabetes, rheumatoid arthritis and Alzheimer's disease, was also shown in vitro and confirmed by the in vivo text on Galleria mellonella. Overall, the obtained results confirm once again the health potential of strawberries; however, the efficacy is dependent on high quality products submitted to correct processing flow charts.

Discovery of novel antituberculosis agents among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives targeting aminoacyl-tRNA synthetases.

Antibiotic resistance is a major problem of tuberculosis treatment. This provides the stimulus for the search of novel molecular targets and approaches to reduce or forestall resistance emergence in Mycobacterium tuberculosis. Earlier, we discovered a novel small-molecular inhibitor among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazoles targeting simultaneously two enzymes-mycobacterial leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase (MetRS), which are promising molecular targets for antibiotic development. Unfortunately, the identified inhibitor does not reveal antibacterial activity toward M. tuberculosis. This study aims to develop novel aminoacyl-tRNA synthetase inhibitors among this chemical class with antibacterial activity toward resistant strains of M. tuberculosis. We performed molecular docking of the library of 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives and selected 41 compounds for investigation of their inhibitory activity toward MetRS and LeuRS in aminoacylation assay and antibacterial activity toward M. tuberculosis strains using microdilution assay. In vitro screening resulted in 10 compounds active against MetRS and 3 compounds active against LeuRS. Structure-related relationships (SAR) were established. The antibacterial screening revealed 4 compounds active toward M. tuberculosis mono-resistant strains in the range of concentrations 2-20 mg/L. Among these compounds, only one compound 27 has significant enzyme inhibitory activity toward mycobacterial MetRS (IC50 = 148.5 µM). The MIC for this compound toward M. tuberculosis H37Rv strain is 12.5 µM. This compound is not cytotoxic to human HEK293 and HepG2 cell lines. Therefore, 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives can be used for further chemical optimization and biological research to find non-toxic antituberculosis agents with a novel mechanism of action.

Inhibitory activity and mechanism of trilobatin on tyrosinase: kinetics, interaction mechanism and molecular docking.

Tyrosinase is the rate-limiting enzyme controlling the production of melanin, and tyrosinase inhibitors can regulate the overproduction of melanin by inhibiting tyrosinase activity, which is an effective method to treat pigmentation disorders. In this study, kinetic analysis, multispectroscopic methods and molecular simulation were used to investigate the inhibitory activity and mechanism of trilobatin on tyrosinase. The kinetic analysis showed that trilobatin had significant inhibitory activity on tyrosinase in a reversible and mixed-type manner with IC50 values of (2.24 ± 0.35) × 10-5 mol L-1. The intrinsic fluorescence of tyrosinase was quenched by trilobatin through a static quenching mechanism. Different spectroscopic measurements demonstrated that trilobatin could change the microenvironments and conformation of tyrosinase and molecular docking determined the binding site of quercetin on tyrosinase.

Targeting adhesion in fungal pathogen Candida albicans.

Fungal infections with increasing resistance to conventional therapies are a growing concern. Candida albicans is a major opportunistic yeast responsible for mucosal and invasive infections. Targeting the initial step of the infection process (i.e., C. albicans adhesion to the host cell) is a promising strategy. A wide variety of molecules can interfere with adhesion processes via an assortment of mechanisms. Herein, we focus on how small molecules disrupt biosynthesis of fungal cell wall components and membrane structure, prevent the localization of GPI-anchor proteins, inhibit production of enzymes involved in adhesion, downregulate genes encoding adhesins and competitively inhibit receptor interactions. As a result, adhesion of C. albicans to host cells is reduced, paving the way to new classes of antifungal agents.

Expedient Discovery for Novel Antifungal Leads Targeting Succinate Dehydrogenase: Pyrazole-4-formylhydrazide Derivatives Bearing a Diphenyl Ether Fragment.

The pyrazole-4-carboxamide scaffold containing a flexible amide chain has emerged as the molecular skeleton of highly efficient agricultural fungicides targeting succinate dehydrogenase (SDH). Based on the above vital structural features of succinate dehydrogenase inhibitors (SDHI), three types of novel pyrazole-4-formylhydrazine derivatives bearing a diphenyl ether moiety were rationally conceived under the guidance of a virtual docking comparison between bioactive molecules and SDH. Consistent with the virtual verification results of a molecular docking comparison, the in vitro antifungal bioassays indicated that the skeleton structure of title compounds should be optimized as an N'-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide scaffold. Strikingly, N'-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives 11o against Rhizoctonia solani, 11m against Fusarium graminearum, and 11g against Botrytis cinerea exhibited excellent antifungal effects, with corresponding EC50 values of 0.14, 0.27, and 0.52 µg/mL, which were obviously better than carbendazim against R. solani (0.34 µg/mL) and F. graminearum (0.57 µg/mL) as well as penthiopyrad against B. cinerea (0.83 µg/mL). The relative studies on an in vivo bioassay against R. solani, bioactive evaluation against SDH, and molecular docking were further explored to ascertain the practical value of compound 11o as a potential fungicide targeting SDH. The present work provided a non-negligible complement for the structural optimization of antifungal leads targeting SDH.

Deciphering potential inhibitors targeting THI4 of Fusarium solani sp. to combat fungal keratitis: An integrative computational approach.

Mycotic keratitis is a fungal infection of corneal epithelium. It is more common in tropical and subtropical countries and is one of the leading causes of blindness. Many of the antifungal drugs have been less effective in treating this condition and certain drugs which are efficient and yet limited in use due to its extreme side effects. Hence, in this study an attempt is made to identify potential and least toxic antifungal inhibitors that targets thiamine thiazole synthase, a novel target for suppressing Fusarium solani subsp.pisi (Nectria haematococca MPVI) infections, to combat mycotic keratitis. Integrative computational approaches involving model refinement, molecular dynamics simulation and High throughput virtual screening (HTVS) were applied through integrative multi precision mode in order to identify potential inhibitors. Moreover, machine learning approach was also implemented to prioritize potential inhibitors that are ophthalmic adaptive, as well as antifungal molecule. From the NCI and Maybridge datasets, for HTVS only 209,872 compounds that surpassed ligand property filtration were considered, which resulted in 209 compounds after XP docking. Among the top 5 compounds from XP docking, on cumulative analysis only 2-(1-hydroxyethyl)-1,3-thiazole-4-carboxamide was prioritized as the most potential hit, as it showed higher order of significance in terms of binding affinity, structural stability and therapeutic relevance for the treatment of Mycotic keratitis. Thus, widening the scope for novel antifungal therapy in ophthalmic infections.

An Antivirulence Approach for Preventing Cryptococcus neoformans from Crossing the Blood-Brain Barrier via Novel Natural Product Inhibitors of a Fungal Metalloprotease.

Cryptococcus neoformans (Cn) is the leading cause of fungal meningitis, a deadly disease with limited therapeutic options. Dissemination to the central nervous system hinges on the ability of Cn to breach the blood-brain barrier (BBB) and is considered an attribute of Cn virulence. Targeting virulence instead of growth for antifungal drug development has not been fully exploited despite the benefits of this approach. Mpr1 is a secreted fungal metalloprotease not required for fungal growth, but rather, it functions as a virulence factor by facilitating Cn migration across the BBB. This central role for Mpr1, its extracellular location, and lack of expression in mammalian cells make Mpr1 a high-value target for an antivirulence approach aimed at developing therapeutics for cryptococcal meningitis. To test this notion, we devised a large-scale screen to identify compounds that prohibited Cn from crossing the BBB by selectively blocking Mpr1 proteolytic activity, without inhibiting the growth of Cn A phytochemical natural product-derived library was screened to identify new molecular scaffolds of prototypes unique to a Cn microecosystem. Of the 240 pure natural products examined, 3 lead compounds, abietic acid, diosgenin, and lupinine inhibited Mpr1 proteolytic activity with 50% inhibitory concentration (IC50) values of <10 µM, displayed little to no mammalian cell toxicity, and did not affect Cn growth. Notably, the lead compounds blocked Cn from crossing the BBB, without damaging the barrier integrity, suggesting the bioactive molecules had no off-target effects. We propose that these new drug scaffolds are promising candidates for the development of antivirulence therapy against cryptococcal meningitis.IMPORTANCE Fungal infections like cryptococcal meningitis are difficult to resolve because of the limited therapies available. The small arsenal of antifungal drugs reflect the difficulty in finding available targets in fungi because like mammalian cells, fungi are eukaryotes. The limited efficacy, toxicity, and rising resistance of antifungals contribute to the high morbidity and mortality of fungal infections and further underscore the dire but unmet need for new antifungal drugs. The traditional approach in antifungal drug development has been to target fungal growth, but an attractive alternative is to target mechanisms of pathogenesis. An important attribute of Cryptococcus neoformans (Cn) pathogenesis is its ability to enter the central nervous system. Here, we describe a large-scale screen that identified three natural products that prevented Cn from crossing the blood-brain barrier by inhibiting the virulence factor Mpr1 without affecting the growth of Cn We propose that compounds identified here could be further developed as antivirulence therapy that would be administered preemptively or serve as a prophylactic in patients at high risk for developing cryptococcal meningitis.

Dihydrofolate Reductase Is a Valid Target for Antifungal Development in the Human Pathogen Candida albicans.

While the folate biosynthetic pathway has provided a rich source of antibacterial, antiprotozoal, and anticancer therapies, it has not yet been exploited to develop uniquely antifungal agents. Although there have been attempts to develop fungal-specific inhibitors of dihydrofolate reductase (DHFR), the protein itself has not been unequivocally validated as essential for fungal growth or virulence. The purpose of this study was to establish dihydrofolate reductase as a valid antifungal target. Using a strain with doxycycline-repressible transcription of DFR1 (PTETO-DFR1 strain), we were able to demonstrate that Dfr1p is essential for growth in vitro Furthermore, nutritional supplements of most forms of folate are not sufficient to restore growth when Dfr1p expression is suppressed or when its activity is directly inhibited by methotrexate, indicating that Candida albicans has a limited capacity to acquire or utilize exogenous sources of folate. Finally, the PTETO-DFR1 strain was rendered avirulent in a mouse model of disseminated candidiasis upon doxycycline treatment. Collectively, these results confirm the validity of targeting dihydrofolate reductase and, by inference, other enzymes in the folate biosynthetic pathway as a strategy to devise new and efficacious therapies to combat life-threatening invasive fungal infections.IMPORTANCE The folate biosynthetic pathway is a promising and understudied source for novel antifungals. Even dihydrofolate reductase (DHFR), a well-characterized and historically important drug target, has not been conclusively validated as an antifungal target. Here, we demonstrate that repression of DHFR inhibits growth of Candida albicans, a major human fungal pathogen. Methotrexate, an antifolate, also inhibits growth but through pH-dependent activity. In addition, we show that C. albicans has a limited ability to take up or utilize exogenous folates as only the addition of high concentrations of folinic acid restored growth in the presence of methotrexate. Finally, we show that repression of DHFR in a mouse model of infection was sufficient to eliminate host mortality. Our work conclusively establishes DHFR as a valid antifungal target in C. albicans.

The discovery of potential phosphopantetheinyl transferase Ppt2 inhibitors against drug-resistant Candida albicans.

With the high-frequency use or abuse of antifungal drugs, the crisis of drug-resistant fungi continues to increase worldwide; in particular, the infection of drug-resistant Candida albicans brings the great challenge to the clinical treatment. Therefore, to decelerate the spread of this resistance, it is extremely urgent to facilitate the new antifungal targets with novel drugs. Phosphopantetheinyl transferases PPTases (Ppt2 in Candida albicans) had been identified in bacterium and fungi and mammals, effects as a vital enzyme in the metabolism of organisms in C. albicans. Ppt2 transfers the phosphopantetheinyl group of coenzyme A to the acyl carrier protein Acp1 in mitochondria for the synthesis of lipoic acid that is essential for fungal respiration, so making Ppt2 an ideal target for antifungal drugs. In this study, 110 FDA-approved drugs were utilized to investigate the Ppt2 inhibition against drug-resistant Candida albicans by the improved fluorescence polarization experiments, which have enough druggability and structural variety under the novel strategy of drug repurposing. Thereinto, eight agents revealed the favourable Ppt2 inhibitory activities. Further, broth microdilution assay of incubating C. albicans with these eight drugs showed that pterostilbene, procyanidine, dichlorophen and tea polyphenol had the superior MIC values. In summary, these findings provide more valuable insight into the treatment of drug-resistant C. albicans.