Discovery Of New Antifungal Agents Research Articles

Rational design, synthesis, analysis and antifungal activity of novel myristic acid derivatives as N-myristoyltransferase inhibitors

A demand for the discovery of new antifungal agents has rose significantly because of a rise in morbidity and related mortality due to systemic fungal infections. N-myristoyltransferase (NMT) has been recognized as a new target for the treatment of fungi infections in recent years. Herein, we report a new series of myristic acid derivatives as antifungal agents by inhibiting N-myristoyltransferase (NMT). The myristic acid derivatives were rationally designed by targeting NMT via docking studies and ADMET studies. The designed myristic acid derivatives were prepared by converting myristic acid to acid chloride followed by coupling with aryl amines to yield the title compounds. The synthesized compounds were purified and analyzed by IR, NMR and Mass spectral analysis. Thereafter, we perform the target NMT inhibition assay. In NMT inhibition assay results, the compound 3g (IC50 = 0.931 μM) had superior activity than other myristic acid derivatives and myristic acid (IC50 = 4.213 μM). In vitro antifungal screening activity results, the compound 3g was more active against Candida albicans (MIC50 =10.94 μg mL−1) than fluconazole (MIC50 = 17.76 μg mL−1). In case of Aspergillus niger (A. nig) strain, compound 3b (MIC50 =13.17 μg mL−1) shows better activity than fluconazole (MIC50 =16.30 μg mL−1). The illustration about the design, synthesis, analysis, and evaluation of myristic acid derivatives is reported.

Antifungal activity of 2-chloro-N-phenylacetamide: a new molecule with fungicidal and antibiofilm activity against fluconazole-resistant Candida spp.

In the current context of emerging drug-resistant fungal pathogens such as Candida albicans and Candida parapsilosis, discovery of new antifungal agents is an urgent matter. This research aimed to evaluate the antifungal potential of 2-chloro-N-phenylacetamide against fluconazole-resistant clinical strains of C. albicans and C. parapsilosis. The antifungal activity of 2-chloro-N-phenylacetamide was evaluated in vitro by the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), inhibition of biofilm formation and its rupture, sorbitol and ergosterol assays, and association between this molecule and common antifungal drugs, amphotericin B and fluconazole. The test product inhibited all strains of C. albicans and C. parapsilosis, with a MIC ranging from 128 to 256 µg.mL-1, and a MFC of 512-1,024 µg.mL-1. It also inhibited up to 92% of biofilm formation and rupture of up to 87% of preformed biofilm. 2-chloro-N-phenylacetamide did not promote antifungal activity through binding to cellular membrane ergosterol nor it damages the fungal cell wall. Antagonism was observed when combining this substance with amphotericin B and fluconazole. The substance exhibited significant antifungal activity by inhibiting both planktonic cells and biofilm of fluconazole-resistant strains. Its combination with other antifungals should be avoided and its mechanism of action remains to be established.

Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens.

Fungal phytopathogens are the major agents responsible for causing severe damage to and losses in agricultural crops worldwide. Botrytis cinerea, Colletotrichum acutatum, Fusarium proliferatum, and Magnaporthe grisea are included in the top ten fungal phytopathogens that impose important plant diseases on a broad range of crops. Microbial natural products can be an attractive alternative for the biological control of phytopathogens. The objective of this work was to develop and validate a High-throughput Screening (HTS) platform to evaluate the antifungal potential of chemicals and natural products against these four important plant pathogens. Several experiments were performed to establish the optimal assay conditions that provide the best reproducibility and robustness. For this purpose, we have evaluated two media formulations (SDB and RPMI-1640), several inoculum concentrations (1 × 106, 5 × 105 and 5 × 106 conidia/mL), the germination curves for each strain, each strain's tolerance to dimethyl sulfoxide (DMSO), and the Dose Response Curves (DRC) of the antifungal control (Amphotericin B). The assays were performed in 96-well plate format, where absorbance at 620 nm was measured before and after incubation to evaluate growth inhibition, and fluorescence intensity at 570 nm excitation and 615 nm emission was monitored after resazurin addition for cell viability evaluation. Quality control parameters (RZ' Factors and Signal to Background (S/B) ratios) were determined for each assay batch. The assay conditions were finally validated by titrating 40 known relevant antifungal agents and testing 2400 microbial natural product extracts from the MEDINA Library through both HTS agar-based and HTS microdilution-based set-ups on the four phytopathogens.

ETHYL ACETATE FRACTION IN ETHANOL EXTRACT OF NONI FRUIT MODIFIED BY ZEOLITE AS ANTI-SEBORRHEIC DERMATITIS: In-vitro AND In-silico STUDIES

Seborrheic dermatitis caused by Malassezia globosa is a relapsing disease that allows for multi-drug resistance. The discovery of new antifungal agents and zeolites as a carrier is needed for further antifungal treatment. This research aims to determine the antifungal activity from extracts and fractions of Noni fruits with or without activated zeolite and predict its inhibitory activity against LIP1(SMG1) enzyme by in silico test. According to statistical results, the extract 20%-zeolite and ethyl acetate fraction 5%-zeolite of Noni fruits provided a higher inhibition zone than those without zeolite. Ethyl acetate fractions as active compounds identified by GC-MS followed by molecular docking. The prediction of the molecular docking depicts that bromocriptine (alkaloid quinoline group) showed a lower binding affinity (∆G) compared to ketoconazole as a standard with similar amino acid residues Asn253, Phe278, Gly291, and Gln282. The SEM-EDX description of ethyl acetate fraction modified by zeolite shows that the active compounds from Noni fruits adsorbed the zeolite framework well. ADMET-Tox properties of bromocriptine were neither mutagenic nor carcinogenic. Alkaloid group is effective as a new antifungal agent for Seborrheic dermatitis, and zeolite as a carrier matrix provides a better inhibitory activity.

Biocontrol Potential of Aspergillus Species Producing Antimicrobial Metabolites.

Microbial metabolites have been recognized as an important source for the discovery of new antifungal agents because of their diverse chemical structures with novel modes of action. In the course of our screening for new antifungal agents from microbes, we found that culture filtrates of two fungal species Aspergillus candidus SFC20200425-M11 and Aspergillus montenegroi SFC20200425-M27 have the potentials to reduce the development of fungal plant diseases such as tomato late blight and wheat leaf rust. From these two Aspergillus spp., we isolated a total of seven active compounds, including two new compounds (4 and 6), and identified their chemical structures based on the NMR spectral analyses: sphaeropsidin A (1), (R)-formosusin A (2), (R)-variotin (3), candidusin (4), asperlin (5), montenegrol (6), and protulactone A (7). Based on the results of the in vitro bioassays of 11 plant pathogenic fungi and bacteria, sphaeropsidin A (1), (R)-formosusin A (2), (R)-variotin (3), and asperlin (5) exhibited a wide range of antimicrobial activity. Furthermore, when plants were treated with sphaeropsidin A (1) and (R)-formosusin A (2) at a concentration of 500 μg/ml, sphaeropsidin A (1) exhibited an efficacy disease control value of 96 and 90% compared to non-treated control against tomato late blight and wheat leaf rust, and (R)-formosusin A (2) strongly reduced the development of tomato gray mold by 82%. Asperlin (5) at a concentration of 500 μg/ml effectively controlled the development of tomato late blight and wheat leaf rust with a disease control value of 95%. Given that culture filtrates and active compounds derived from two Aspergillus spp. exhibited disease control efficacies, our results suggest that the Aspergillus-produced antifungal compounds could be useful for the development of new natural fungicides.

Advances in fungal chemical genomics for the discovery of new antifungal agents.

Invasive fungal infections have escalated from a rare curiosity to a major cause of human mortality around the globe. This is in part due to a scarcity in the number of antifungal drugs available to combat mycotic disease, making the discovery of novel bioactive compounds and determining their mode of action of utmost importance. The development and application of chemical genomic assays using the model yeast Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of diverse molecules in a living cell. Furthermore, complementary assays are continually being developed in fungal pathogens, most notably Candida albicans and Cryptococcus neoformans, to elucidate compound mechanism of action directly in the pathogen of interest. Collectively, the suite of chemical genetic assays that have been developed in multiple fungal species enables the identification of candidate drug target genes, as well as genes involved in buffering drug target pathways, and genes involved in general cellular responses to small molecules. In this review, we examine current yeast chemical genomic assays and highlight how such resources provide powerful tools that can be utilized to bolster the antifungal pipeline.

LEGO‐Inspired Drug Design: Unveiling a Class of Benzo[d]thiazoles Containing a 3,4‐Dihydroxyphenyl Moiety as Plasma Membrane H+‐ATPase Inhibitors

The fungal plasma membrane H+ -ATPase (Pma1p) is a potential target for the discovery of new antifungal agents. Surprisingly, no structure-activity relationship studies for small molecules targeting Pma1p have been reported. Herein, we disclose a LEGO-inspired fragment assembly strategy for the design, synthesis, and discovery of benzo[d]thiazoles containing a 3,4-dihydroxyphenyl moiety as potential Pma1p inhibitors. A series of 2-(benzo[d]thiazol-2-ylthio)-1-(3,4-dihydroxyphenyl)ethanones was found to inhibit Pma1p, with the most potent IC50 value of 8 μm in an in vitro plasma membrane H+ -ATPase assay. These compounds were also found to strongly inhibit the action of proton pumping when Pma1p was reconstituted into liposomes. 1-(3,4-Dihydroxyphenyl)-2-((6-(trifluoromethyl)benzo[d]thiazol-2-yl)thio)ethan-1-one (compound 38) showed inhibitory activities on the growth of Candida albicans and Saccharomyces cerevisiae, which could be correlated and substantiated with the ability to inhibit Pma1p in vitro.

Isolation and antifungal screening of endophytic fungi from Erigeron canadensis

Sixteen fungal strains isolated from the Erigeron canadensis, one of traditional Chinese medicines used to treat the pathogenic infection and dysentery, were evaluated for their antifungal activities against one human pathogen Candida albicans, and two phytopathogens, Colletotrichum fructicola and Rhizoctonia cerealis. The bioassay results indicated that the ethyl acetate extract of the fermentation broth of these fungal endophytes had stronger antimicrobial activities. Among these endophytic strains, the ethyl acetate extracts of strains NPR003 and NPR005 showed the strongest inhibitory effects and has potential application in the discovery of new antifungal agents. This was the first report on the isolation of endophytic fungi from E. canadensis and evaluation of their antifungal activities.
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Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani.

Many Fusarium species are able to cause severe infections in plants as well as in animals and humans. Therefore, the discovery of new antifungal agents is of paramount importance. CaThi belongs to the thionins, which are cationic peptides with low molecular weights (∼5 kDa) that have toxic effects against various microorganisms. Herein, we study the mechanism of action of CaThi and its combinatory effect with fluconazole (FLC) against Fusarium solani. The mechanism of action of CaThi was studied by growth inhibition, viability, plasma membrane permeabilization, ROS induction, caspase activation, localization, and DNA binding capability, as assessed with Sytox green, DAB, FITC-VAD-FMK, CaThi-FITC, and gel shift assays. The combinatory effect of CaThi and FLC was assessed using a growth inhibition assay. Our results demonstrated that CaThi present a dose dependent activity and at the higher used concentration (50 µgmL-1 ) inhibits 83% of F. solani growth, prevents the formation of hyphae, permeabilizes membranes, induces endogenous H2 O2 , activates caspases, and localizes intracellularly. CaThi combined with FLC, at concentrations that alone do not inhibit F. solani, result in 100% death of F. solani when combined. The data presented in this study demonstrate that CaThi causes death of F. solani via apoptosis; an intracellular target may also be involved. Combined treatment using CaThi and FLC is a strong candidate for studies aimed at improved targeting of F. solani. This strategy is of particular interest because it minimizes selection of resistant microorganisms.

Anti-Candida Activity of New Azole Derivatives Alone and in Combination with Fluconazole.

The rate of Candida spp. infection is increasing, and resistance to azole antifungals is becoming increasingly common. Therefore, there is a need for discovery of new antifungal agents and for development of new modes of treatment using existing agents. In this in vitro study, the antifungal activity of two new imidazole derivatives was tested against a set of 20 Candida isolates, which included five different species. Treatment was carried out with the compound alone and in combination with fluconazole. Overall, we found that one of the new compounds, 31, was similar to fluconazole (FLC) in its efficacy against the Candida isolates and that compound 42 was superior to FLC. Furthermore, when combined with FLC, both compounds showed synergistic effects against 17 of the 20 tested isolates. No antagonistic interactions were observed. This study shows that our two new imidazole-derived compounds have good potential as general treatments for Candida infection and as a means to improve the current treatments with FLC.

Homology modeling of lanosterol 14α-demethylase of Candida albicans and insights into azole binding

Candida albicans is one of the most common causes of invasive fungal infections. Azole antifungal agents are widely used as fungal antibiotics which inhibit the cytochrome P450 sterol 14α-demethylase (CYP51). This study describes the homology modeling and three-dimensional model of CYP51 from C. albicans SC5314. Dope score, GA341 of Modeller, PROCHECK statistics, Ramachandran, and G-factors were used to analyze and evaluate a final model. Molecular docking identified the binding mode of the azole antifungal agents with modeled CYP51. The obtained results of Autodock program, compounds 1–4, were determined the heterocyclic nitrogen atom of azole bound to the heme iron atom in the binding site of the enzyme. Based on the results of this study, we conclude that the structural model of C. albicans SC5314 can be used in azole optimization, virtual screening and de novo inhibitor design for the discovery of new antifungal agents. This study describes the homology modeling and three-dimensional model of CYP51 from C. albicans SC5314. Dope score and GA341 of Modeller, and PROCHECK statistics, Ramachandran, and G-factors were used to analysis and evaluate of the final model.

Ethnobotanical survey of medicinal plants used in the management of opportunistic fungal infections in HIV/AIDS patients in the Amathole District of the Eastern Cape Province, South Africa

An estimated 5.7 million people were living with HIV and AIDS in South Africa in 2009, more than in any other country. Up to 90% of all HIV/AIDS patients develop opportunistic fungi infections (OFIs) at some point during the course of the disease and 10 to 20% dies as a direct consequence of these. Despite the broad use of medicinal plants in South Africa, there is a dearth of knowledge regarding the use of such plants in the management of opportunistic fungal infections in HIV/AIDS patients. Knowledge of these plants is very important as this can serve as leads in the discovery of new antifungal agents. The purpose of this study was to document the indigenous knowledge of medicinal plants used in the management of opportunistic fungal infections in HIV/AIDS patients in the Eastern Cape, South Africa. Information was obtained through conversations with traditional healers and AIDS patients with the aid of semi-structured questionnaires, direct observations and by reviewing studies reported in the literature. A total of 123 informants participated in the study, including 22 (21.8%) traditional healers and 101 (78.2%) HIV/AIDS patients. Thirty two plant species distributed in 26 families and 32 genera were identified as being used to treat one or more of the OFIs. Considering the widespread use of these medicinal plants to treat various ailments and their current use in the management of oportunistic fungal infections in HIV/AIDS patients, it therefore becomes crucial to scientifically validate the therapeutic uses and safety of these plants through phytochemical screening, antifungal susceptibility tests and toxicological studies. Key words: Medicinal plants, opportunistic fungal infections, human immunodeficiency virus/acquired immunodeficiency syndromes (HIV/AIDS), ethnobotanical survey, Eastern Cape Province, South Africa.

Antifungal Activity of the Essential Oils of Callitris neocaledonica and C. sulcata Heartwood (Cupressaceae)

Mortality due to fungal infections has increased substantially, becoming a worldwide problem in public health. As a contribution to the discovery of new antifungal agents, the properties of the heartwood essential oils of two trees growing in New Caledonia, Callitris neocaledonica and C. sulcata (Cupressaceae) were investigated. The essential oils extracted by hydrodistillation were characterized by GC-FID and GC/MS analyses. From C. neocaledonica oil, 31 constituents were identified, representing 97.0% of the total oil composition, which was mainly constituted by oxygenated sesquiterpenes (88%). Among them, guaiol (1; 30.2%), bulnesol (2; 12.5%), α-eudesmol (3; 10.5%), β-eudesmol (4; 10.5%), γ-eudesmol (10.2%), and elemol (4.9%) predominated. The chemical composition of C. sulcata oil, from which 39 constituents were identified (96.8% of the total oil composition), showed some similarities with that of C. neocaledonica oil. The major constituents were also oxygenated sesquiterpenes, accounting for 78.5% of the oil, amongst them, mainly compounds 1 (16.1%), 3 and 4 (9.7% each), as well as 2 (7.4%). The antifungal activity of the oils against clinical isolates of four dermatophytic fungi (Trichophyton mentagrophytes, T. rubrum, Microsporum canis, and M. gypseum) and six yeasts (Candida albicans, C. parapsilosis, C. glabrata, C. krusei, Cryptococcus neoformans, and Cryptococcus gattii) was tested by determining minimum inhibitory concentrations (MICs) using the microdilution method. The best antifungal activities of the C. neocaledonica and C. sulcata oils were obtained against C. krusei (MICs of 3.9 and 0.975 μg/ml, resp.). These MIC values were similar to those of the reference drugs itraconazole and fluconazole (1.0 and 0.5 mg/ml, resp.). The oils were also subjected to a screening for their possible DPPH(.) (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity. C. neocaledonica essential oil was more active than C. sulcata oil (93.3 vs. 32.2% DPPH(.) scavenged at 250 μg/ml).

Traditional use of medicinal plants for anti-ringworm therapy in some parts of Kamrup District of Assam, a North Eastern State of India

ObjectiveTo survey traditional use of medicinal plants for anti-ringworm therapy from December 2007 to January 2009 in some parts of Kamrup district of Assam, a North Eastern State of India. MethodsBased on a standard questionnaire, local inhabitants were interviewed for their knowledge of medicinal plants used against ringworm. Each entry includes the botanical and prevalent local name(s), part(s) used, and mode of use against the diseases. ResultsThis report deals with 46 plant species belonging to 30 different families used as herbal remedies for the treatment of Dermatophytosis. ConclusionsThe study reveals several medicinal plants used by the local people for the treatment of cutaneous infections caused by dermatophytes. Such study may lead to the discovery of new antifungal agents from natural sources.

Antifúngicos disponibles para el tratamiento de las micosis ungueales

Nail fungal infections are considered one of the major dermatological problems due to their high rate of therapeutic failure, management and treatment difficulties. Long-term treatments, inadequate therapies, mycological misdiagnosis and follow-up, secondary alterations of the nail, and resistant microorganisms, are some of the causes of these complications. Although the discovery of new antifungal agents has provided some effective molecules, none of the current available drugs are totally effective. It is important to continue researching in this field to provide new antifungal agents and combined therapies.

Toward the discovery of new antifungal agents: The design and validation of a novel 2'P-RNA probe and high throughput screening assay against 2'-phosphotransferase Tpt1p

We report the solid-phase synthesis of novel 2'P-RNA probes for use in fluorescence polarization (FP) ligand binding assays that screens for inhibitors of the yeast 2'- phosphotransferase Tpt1p. The probe was synthesized by utilizing silyl phosphoramidite chemistry and a phosphoramidite synthon containing an orthogonal (DMT) protecting group at its 2'-position. Regioselective removal of the 2'-DMT group and phosphitylation of the unmasked 2'-hydroxyl group afforded the desired 2'P-RNA sequence.