PHYTOCHROME-INTERACTING FACTOR 4/HEMERA-mediated thermosensory growth requires the Mediator subunit MED14.

While moderately elevated ambient temperatures do not trigger stress responses in plants, they do substantially stimulate the growth of specific organs through a process known as thermomorphogenesis. The basic helix-loop-helix transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) plays a central role in regulating thermomorphogenetic hypocotyl elongation in various plant species, including Arabidopsis (Arabidopsis thaliana). Although it is well known that PIF4 and its co-activator HEMERA (HMR) promote plant thermosensory growth by activating genes involved in the biosynthesis and signaling of the phytohormone auxin, the detailed molecular mechanism of such transcriptional activation is not clear. In this report, we investigated the role of the Mediator complex in the PIF4/HMR-mediated thermoresponsive gene expression. Through the characterization of various mutants of the Mediator complex, a tail subunit named MED14 was identified as an essential factor for thermomorphogenetic hypocotyl growth. MED14 was required for the thermal induction of PIF4 target genes but had a marginal effect on the levels of PIF4 and HMR. Further transcriptomic analyses confirmed that the expression of numerous PIF4/HMR-dependent, auxin-related genes required MED14 at warm temperatures. Moreover, PIF4 and HMR physically interacted with MED14 and both were indispensable for the association of MED14 with the promoters of these thermoresponsive genes. While PIF4 did not regulate MED14 levels, HMR was required for the transcript abundance of MED14. Taken together, these results unveil an important thermomorphogenetic mechanism, in which PIF4 and HMR recruit the Mediator complex to activate auxin-related growth-promoting genes when plants sense moderate increases in ambient temperature.

The Oligostilbene Gnetin H Is a Novel Glycolysis Inhibitor That Regulates Thioredoxin Interacting Protein Expression and Synergizes with OXPHOS Inhibitor in Cancer Cells.

Since aerobic glycolysis was first observed in tumors almost a century ago by Otto Warburg, the field of cancer cell metabolism has sparked the interest of scientists around the world as it might offer new avenues of treatment for malignant cells. Our current study claims the discovery of gnetin H (GH) as a novel glycolysis inhibitor that can decrease metabolic activity and lactic acid synthesis and displays a strong cytostatic effect in melanoma and glioblastoma cells. Compared to most of the other glycolysis inhibitors used in combination with the complex-1 mitochondrial inhibitor phenformin (Phen), GH more potently inhibited cell growth. RNA-Seq with the T98G glioblastoma cell line treated with GH showed more than an 80-fold reduction in thioredoxin interacting protein (TXNIP) expression, indicating that GH has a direct effect on regulating a key gene involved in the homeostasis of cellular glucose. GH in combination with phenformin also substantially enhances the levels of p-AMPK, a marker of metabolic catastrophe. These findings suggest that the concurrent use of the glycolytic inhibitor GH with a complex-1 mitochondrial inhibitor could be used as a powerful tool for inducing metabolic catastrophe in cancer cells and reducing their growth.

Structure and Antimicrobial Activity of Rare Lactone Lipids from the Sooty Mold (Scorias spongiosa).

Two new lactone lipids, scoriosin (1) and its methyl ester (2), with a rare furylidene ring joined to a tetrahydrofurandione ring, were isolated from Scorias spongiosa, commonly referred to as sooty mold. The planar structure of these compounds was assigned by 1D and 2D NMR. The conformational analysis of these molecules was undertaken to evaluate the relative and absolute configuration through GIAO NMR chemical shift analysis and ECD calculation. In addition to the potent antimicrobial activities, compound 2 strongly potentiated the activity of amphotericin B against Cryptococcus neoformans, suggesting the potential utility of this compound in combination therapies for treating cryptococcal infections.

Synthesis and Antifungal Activity Evaluation of Phloeodictine Analogues.

The phloeodictine-based 6-hydroxy-2,3,4,6-tetrahydropyrrolo[1,2-a]pyrimidinium structural moiety with an n-tetradecyl side chain at C-6 has been demonstrated to be a new antifungal template. Thirty-four new synthetic analogues with modifications of the bicyclic tetrahydropyrrolopyrimidinium skeleton and the N-1 side chain have been prepared and evaluated for in vitro antifungal activities against the clinically important fungal pathogens including Cryptococcus neoformans ATCC 90113, Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, and Aspergillus fumigatus ATCC 90906. Nineteen compounds (5, 21-31, 34-38, 44, and 48) showed antifungal activities against the aforementioned five fungal pathogens with minimum inhibitory concentrations (MICs) in the range 0.88-10 µM, and all were fungicidal with minimum fungicidal concentrations (MFCs) similar to the respective MIC values. Compounds 24, 36, and 48 were especially active against C. neoformans ATCC 90113 with MIC/MFC values of 1.0/1.0, 1.6/1.6, and 1.3/2.0 µM but exhibited low cytotoxicity with an IC50 > 40 µM against the mammalian Vero cells. The structure and antifungal activity relationship indicates that synthetic modifications of the phloeodictines can afford analogues with potent antifungal activity and reduced cytotoxicity, necessitating further preclinical studies of this new class of antifungal compounds.

Two plant-derived aporphinoid alkaloids exert their antifungal activity by disrupting mitochondrial iron-sulfur cluster biosynthesis.

Eupolauridine and liriodenine are plant-derived aporphinoid alkaloids that exhibit potent inhibitory activity against the opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans However, the molecular mechanism of this antifungal activity is unknown. In this study, we show that eupolauridine 9591 (E9591), a synthetic analog of eupolauridine, and liriodenine methiodide (LMT), a methiodide salt of liriodenine, mediate their antifungal activities by disrupting mitochondrial iron-sulfur (Fe-S) cluster synthesis. Several lines of evidence supported this conclusion. First, both E9591 and LMT elicited a transcriptional response indicative of iron imbalance, causing the induction of genes that are required for iron uptake and for the maintenance of cellular iron homeostasis. Second, a genome-wide fitness profile analysis showed that yeast mutants with deletions in iron homeostasis-related genes were hypersensitive to E9591 and LMT. Third, treatment of wild-type yeast cells with E9591 or LMT generated cellular defects that mimicked deficiencies in mitochondrial Fe-S cluster synthesis including an increase in mitochondrial iron levels, a decrease in the activities of Fe-S cluster enzymes, a decrease in respiratory function, and an increase in oxidative stress. Collectively, our results demonstrate that E9591 and LMT perturb mitochondrial Fe-S cluster biosynthesis; thus, these two compounds target a cellular pathway that is distinct from the pathways commonly targeted by clinically used antifungal drugs. Therefore, the identification of this pathway as a target for antifungal compounds has potential applications in the development of new antifungal therapies.

Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms.

In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans, grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations.

Synthesis of Natural Acylphloroglucinol-Based Antifungal Compounds against Cryptococcus Species.

Thirty-three natural-product-based acylphloroglucinol derivatives were synthesized to identify antifungal compounds against Cryptococcus spp. that cause the life-threatening disseminated cryptococcosis. In vitro antifungal testing showed that 17 compounds were active against C. neoformans ATCC 90113, C. neoformans H99, and C. gattii ATCC 32609, with minimum inhibitory concentrations (MICs) in the range 1.0-16.7 µg/mL. Analysis of the structure and antifungal activity of these compounds indicated that the 2,4-diacyl- and 2-acyl-4-alkylphloroglucinols were more active than O-alkyl-acylphloroglucinols. The most promising compound found was 2-methyl-1-(2,4,6-trihydroxy-3-(4-isopropylbenzyl)phenyl)propan-1-one (11j), which exhibited potent antifungal activity (MICs, 1.5-2.1 µg/mL) and low cytotoxicity against the mammalian Vero and LLC-PK1 cell lines (IC50 values >50 µg/mL). This compound may serve as a template for further synthesis of new analogues with improved antifungal activity. The findings of the present work may contribute to future antifungal discovery toward pharmaceutical development of new treatments for cryptococcosis.

Sampangine (a Copyrine Alkaloid) Exerts Biological Activities through Cellular Redox Cycling of Its Quinone and Semiquinone Intermediates.

The cananga tree alkaloid sampangine (1) has been extensively investigated for its antimicrobial and antitumor potential. Mechanistic studies have linked its biological activities to the reduction of cellular oxygen, the induction of reactive oxygen species (ROS), and alterations in heme biosynthesis. Based on the yeast gene deletion library screening results that indicated mitochondrial gene deletions enhanced the sensitivity to 1, the effects of 1 on cellular respiration were examined. Sampangine increased oxygen consumption rates in both yeast and human tumor cells. Mechanistic investigation indicated that 1 may have a modest uncoupling effect, but predominately acts by increasing oxygen consumption independent of mitochondrial complex IV. Sampangine thus appears to undergo redox cycling that may involve respiratory chain-dependent reduction to a semi-iminoquinone followed by oxidation and consequent superoxide production. Relatively high concentrations of 1 showed significant neurotoxicity in studies conducted with rat cerebellar granule neurons, indicating that sampangine use may be associated with potential neurotoxicity.

LC-MS- and (1)H NMR Spectroscopy-Guided Identification of Antifungal Diterpenoids from Sagittaria latifolia.

Antifungal screening of small-molecule natural product libraries showed that a column fraction (CF) derived from the plant extract of Sagittaria latifolia was active against the fungal pathogen Cryptococcus neoformans. Dereplication analysis by liquid chromatography-mass spectrometry (LC-MS) and proton nuclear magnetic resonance spectroscopy ((1)H NMR) indicated the presence of new compounds in this CF. Subsequent fractionation of the plant extract resulted in the identification of two new isopimaradiene-type diterpenoids, 1 and 2. The structures of 1 and 2 were determined by chemical methods and spectroscopic analysis as isopimara-7,15-dien-19-ol 19-O-α-l-arabinofuranoside and isopimara-7,15-dien-19-ol 19-O-α-l-(5'-acetoxy)arabinofuranoside, respectively. Compound 1 exhibited IC50 values of 3.7 and 1.8 µg/mL, respectively, against C. neoformans and C. gattii. Its aglycone, isopimara-7,15-dien-19-ol (3), resulting from acid hydrolysis of 1, was also active against the two fungal pathogens, with IC50 values of 9.2 and 6.8 µg/mL, respectively. This study demonstrates that utilization of the combined LC-MS and (1)H NMR analytical tools is an improved chemical screening approach for hit prioritization in natural product drug discovery.

Identification of Botanicals that Unmask ß-Glucan from the Cell Surface of an Opportunistic Fungal Pathogen.

Dectin-1 expressed on host immune cells recognizes ß-glucans within the cell walls of fungal pathogens and plays an important role in the clearance of fungal infections. However, because ß-glucan is masked by an outer layer of mannoproteins, fungal pathogens can evade detection by host immune cells. In this study, a microplate-based screen was developed to identify ß-glucan unmasking activity exhibited by botanicals. This screen measures the activity of a reporter gene in response to the transcriptional activation of NF-κB due to the interaction between ß-glucan on the fungal cell surface and Dectin-1 present on host immune cells. In this proof-of-concept study, we screened a collection of botanicals (10 plants and some of their reported pure compound actives) used in traditional medicine for their antifungal properties. Several hits were identified in samples that unmasked ß-glucan at sub-inhibitory concentrations. The hit samples were confirmed by fluorescent staining with a ß-glucan antibody, verifying that the samples identified in the screen did indeed unmask ß-glucan. These results indicate that the purported antifungal activities attributed to some botanicals may be due, at least in part, to the presence of compounds that exhibit ß-glucan unmasking activity. Enhanced exposure of cell wall ß-glucans would allow the host to build resilience against fungal infections by helping the immune system to detect the pathogen and mount a more effective clearance mechanism. This screen, together with direct killing/growth inhibition assays, may therefore serve as a valuable tool for substantiating the use of botanicals in preventing and/or treating fungal infections.

Synthesis and antifungal activities of miltefosine analogs.

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a-3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5-5.0 µg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5-3.3 µg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections.

A potent plant-derived antifungal acetylenic acid mediates its activity by interfering with fatty acid homeostasis.

6-Nonadecynoic acid (6-NDA), a plant-derived acetylenic acid, exhibits strong inhibitory activity against the human fungal pathogens Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model yeast Saccharomyces cerevisiae to investigate its mechanism of action. 6-NDA elicited a transcriptome response indicative of fatty acid stress, altering the expression of genes that are required for yeast growth in the presence of oleate. Mutants of S. cerevisiae lacking transcription factors that regulate fatty acid ß-oxidation showed increased sensitivity to 6-NDA. Fatty acid profile analysis indicated that 6-NDA inhibited the formation of fatty acids longer than 14 carbons in length. In addition, the growth inhibitory effect of 6-NDA was rescued in the presence of exogenously supplied oleate. To investigate the response of a pathogenic fungal species to 6-NDA, transcriptional profiling and biochemical analyses were also conducted in C. albicans. The transcriptional response and fatty acid profile of C. albicans were comparable to those obtained in S. cerevisiae, and the rescue of growth inhibition with exogenous oleate was also observed in C. albicans. In a fluconazole-resistant clinical isolate of C. albicans, a fungicidal effect was produced when fluconazole was combined with 6-NDA. In hyphal growth assays, 6-NDA inhibited the formation of long hyphal filaments in C. albicans. Collectively, our results indicate that the antifungal activity of 6-NDA is mediated by a disruption in fatty acid homeostasis and that 6-NDA has potential utility in the treatment of superficial Candida infections.

Sampangine inhibits heme biosynthesis in both yeast and human.

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism-by hyperactivating uroporhyrinogen III synthase-to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.

The marine sponge-derived polyketide endoperoxide plakortide F acid mediates its antifungal activity by interfering with calcium homeostasis.

Plakortide F acid (PFA), a marine-derived polyketide endoperoxide, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Saccharomyces cerevisiae to investigate the mechanism of action of this compound. PFA elicited a transcriptome response indicative of a Ca(2+) imbalance, affecting the expression of genes known to be responsive to altered cellular calcium levels. Several additional lines of evidence obtained supported a role for Ca(2+) in PFA's activity. First, mutants lacking calcineurin and various Ca(2+) transporters, including pumps (Pmr1 and Pmc1) and channels (Cch1 and Mid1), showed increased sensitivity to PFA. In addition, the calcineurin inhibitors FK506 and cyclosporine strongly enhanced PFA activity in wild-type cells. Furthermore, PFA activated the transcription of a lacZ reporter gene driven by the calcineurin-dependent response element. Finally, elemental analysis indicated a significant increase in intracellular calcium levels in PFA-treated cells. Collectively, our results demonstrate that PFA mediates its antifungal activity by perturbing Ca(2+) homeostasis, thus representing a potentially novel mechanism distinct from that of currently used antifungal agents.

Puupehenone, a Marine-Sponge-Derived Sesquiterpene Quinone, Potentiates the Antifungal Drug Caspofungin by Disrupting Hsp90 Activity and the Cell Wall Integrity Pathway.

The cell wall-targeting echinocandin antifungals, although potent and well tolerated, are inadequate in treating fungal infections due to their narrow spectrum of activity and their propensity to induce pathogen resistance. A promising strategy to overcome these drawbacks is to combine echinocandins with a molecule that improves their activity and also disrupts drug adaptation pathways. In this study, we show that puupehenone (PUUP), a marine-sponge-derived sesquiterpene quinone, potentiates the echinocandin drug caspofungin (CAS) in CAS-resistant fungal pathogens. We have conducted RNA sequencing (RNA-seq) analysis, followed by genetic and molecular studies, to elucidate PUUP's CAS-potentiating mechanism. We found that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2 (Mpk1), the terminal mitogen-activated protein (MAP) kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its cochaperone Cdc37. Thus, we show that PUUP potentiates CAS activity by a previously undescribed mechanism which involves a disruption of Hsp90 activity and the CWI pathway. Given the requirement of the Hsp90-Cdc37 complex in Slt2 activation, we suggest that inhibitors of this complex would disrupt the CWI pathway and synergize with echinocandins. Therefore, the identification of PUUP's CAS-potentiating mechanism has important implications in the development of new antifungal combination therapies.IMPORTANCE Fungal infections cause more fatalities worldwide each year than malaria or tuberculosis. Currently available antifungal drugs have various limitations, including host toxicity, narrow spectrum of activity, and pathogen resistance. Combining these drugs with small molecules that can overcome these limitations is a useful strategy for extending their clinical use. We have investigated the molecular mechanism by which a marine-derived compound potentiates the activity of the antifungal echinocandin caspofungin. Our findings revealed a mechanism, different from previously reported caspofungin potentiators, in which potentiation is achieved by the disruption of Hsp90 activity and signaling through the cell wall integrity pathway, processes that play important roles in the adaptation to caspofungin in fungal pathogens. Given the importance of stress adaptation in the development of echinocandin resistance, this work will serve as a starting point in the development of new combination therapies that will likely be more effective and less prone to pathogen resistance.

Puupehanol, a sesquiterpene-dihydroquinone derivative from the marine sponge Hyrtios sp.

Puupehanol (1), a new sesquiterpene-dihydroquinone derivative, was isolated from the marine sponge Hyrtios sp., along with the known compounds puupehenone (2) and chloropuupehenone (3) that are responsible for the antifungal activity observed in the extract. The structure of 1 was established as (20R,21R)-21-hydroxy-20,21-dihydropuupehenone by extensive spectroscopic and computational methods. Compound 2 exhibited potent activity against Cryptococcus neoformans and Candida krusei with MFCs of 1.25 and 2.50 microg/mL, respectively.

Verbesinosides A-F, 15,27-cyclooleanane saponins from the American native plant Verbesina virginica.

Verbesinosides A-F (1-6), six new 15,27-cyclooleanane-type triterpenoid saponins carrying different aromatic acyl moieties on the aglycon, were isolated from the leaves and flowers of Verbesina virginica. Their structures were established by interpretation of spectroscopic data and chemical methods. The representative major saponin, verbesinoside A (1), has the structure 21-trimethoxybenzoyl 15alpha,27-cycloolean-12-en-3beta,21beta-diol-28-oic acid 3-O-beta-D-xylopyranosyl(1-->4)-beta-D-xylopyranosyl(1-->2)-beta-D-glucopyranoside. This is the first report of triterpenoid saponins possessing the unique 15,27-cyclooleanane skeleton. The anisotropic effects of the aromatic acyl moieties on the triterpenoid skeleton are discussed.

Role of heme in the antifungal activity of the azaoxoaporphine alkaloid sampangine.

Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling experiments coupled with analyses of mutants were performed in an effort to elucidate its mechanism of action. Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low-oxygen conditions. Several additional lines of evidence obtained suggest that these responses could involve effects on heme. First, the hem1Delta mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine. Furthermore, spectral analyses of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also performed with C. albicans to investigate the response of a pathogenic fungal species to sampangine. Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles, suggestive of heme-related defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme.

FLAVONOL GLYCOSIDES FROM THE NATIVE AMERICAN PLANT GAURA LONGIFLORA.

Phytochemical investigation of the native American plant Gaura longiflora led to the isolation of three new and eight known flavonol glycosides. The structures of the new compounds were established primarily by spectroscopic data as quercetin 3-O-(2″-O-α-l-rhamnopyranosyl-6″-O-E-p-coumaroyl)-ß-D-glucopyranoside (1), kaempferol 3-O-(2″-O-α-l-rhamnopyranosyl-6″-O-E-p-coumaroyl)-ß-d-gluco-pyranoside (2) and quercetin 3-O-(2″-O-α-l-rhamnopyranosyl-6″-O-Z-p-coumaroyl)-ß-D-glucopyrano-side (3).

Potent in vitro antifungal activities of naturally occurring acetylenic acids.

Our continuing effort in antifungal natural product discovery has led to the identification of five 6-acetylenic acids with chain lengths from C(16) to C(20): 6-hexadecynoic acid (compound 1), 6-heptadecynoic acid (compound 2), 6-octadecynoic acid (compound 3), 6-nonadecynoic acid (compound 4), and 6-icosynoic acid (compound 5) from the plant Sommera sabiceoides. Compounds 2 and 5 represent newly isolated fatty acids. The five acetylenic acids were evaluated for their in vitro antifungal activities against Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Trichophyton mentagrophytes, and Trichophyton rubrum by comparison with the positive control drugs amphotericin B, fluconazole, ketoconazole, caspofungin, terbinafine, and undecylenic acid. The compounds showed various degrees of antifungal activity against the 21 tested strains. Compound 4 was the most active, in particular against the dermatophytes T. mentagrophytes and T. rubrum and the opportunistic pathogens C. albicans and A. fumigatus, with MICs comparable to several control drugs. Inclusion of two commercially available acetylenic acids, 9-octadecynoic acid (compound 6) and 5,8,11,14-eicosatetraynoic acid (compound 7), in the in vitro antifungal testing further demonstrated that the antifungal activities of the acetylenic acids were associated with their chain lengths and positional triple bonds. In vitro toxicity testing against mammalian cell lines indicated that compounds 1 to 5 were not toxic at concentrations up to 32 muM. Furthermore, compounds 3 and 4 did not produce obvious toxic effects in mice at a dose of 34 mumol/kg of body weight when administered intraperitoneally. Taking into account the low in vitro and in vivo toxicities and significant antifungal potencies, these 6-acetylenic acids may be excellent leads for further preclinical studies.