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Three specific β(1,3)glucan synthase (GS) inhibitor families, papulacandins, acidic terpenoids, and echinocandins, have been analyzed in Schizosaccharomyces pombe wild-type and papulacandin-resistant cells and GS activities. Papulacandin and enfumafungin produced similar in vivo effects, different from that of echinocandins. Also, papulacandin was the strongest in vitro GS inhibitor (IC(50) 10(3)-10(4)-fold lower than with enfumafungin or pneumocandin), but caspofungin was by far the most efficient antifungal because of the following. 1) It was the only drug that affected resistant cells (minimal inhibitory concentration close to that of the wild type). 2) It was a strong inhibitor of wild-type GS (IC(50) close to that of papulacandin). 3) It was the best inhibitor of mutant GS. Moreover, caspofungin showed a special effect for two GS inhibition activities, of high and low affinity, separated by 2 log orders, with no increase in inhibition. pbr1-8 and pbr1-6 resistances are due to single substitutions in the essential Bgs4 GS, located close to the resistance hot spot 1 region described in Saccharomyces and Candida Fks mutants. Bgs4(pbr)(1-8) contains the E700V change, four residues N-terminal from hot spot 1 defining a larger resistance hot spot 1-1 of 13 amino acids. Bgs4(pbr)(1-6) contains the W760S substitution, defining a new resistance hot spot 1-2. We observed spontaneous revertants of the spherical pbr1-6 phenotype and found that an additional A914V change is involved in the recovery of the wild-type cell shape, but it maintains the resistance phenotype. A better understanding of the mechanism of action of the antifungals available should help to improve their activity and to identify new antifungal targets.

Arborcandin C is a novel antibiotic with potent antifungal activity that inhibits 1,3-beta-glucan synthase in fungi. We examined spontaneous Saccharomyces cerevisiae mutants which are selectively resistant to arborcandin C and revealed that a single amino acid replacement in Fks1p of Asn(470) with Lys or of Leu(642) with Ser confers selective resistance on Fks1p mutants.

The dimorphic fungus Candida albicans is an opportunistic human pathogen. Candidiasis is usually treated with azole antifungal agents. However clinical treatments may fail due to the appearance of resistance to this class of antifungal agents in Candida. Echinocandin derivatives are an alternative for the treatment of these fungal infections and are active against azole resistant isolates of C. albicans. Azoles inhibit the lanosterol 14 alpha demethylase which is a key enzyme in the synthesis of ergosterol. In contrast, the echinocandin class of antibiotics inhibit noncompetitively beta-(1,3)-D-glucan synthesis in vitro. We have investigated the impact of mulundocandin on the proteome of C. albicans and compared it to those of a mulundocandin derivative, as well as to two azoles of different structure, fluconazole and itraconazole. The changes in gene expression underlying the antifungal responses were analyzed by comparative 2-D PAGE. Dose dependant responses were kinetically studied on C. albicans grown at 25 degrees C (yeast form) in synthetic dextrose medium. This study shows that antifungals with a common mechanism of action lead to comparable effects at the proteome level and that a proteomics approach can be used to distinguish different antifungals, with the promise to become a useful tool to study drugs of unknown mechanism of action.

Infection by many fungi activates a variety of calcium dependent defenses in the hosts, slowing or suppressing the attacker and limiting the efficacy of mycoherbicides. The calcium requirement for fungal growth is so low that it could only be implied based on fungi containing calcium-dependent signaling enzymes. Analytical grade media contain <2 microM calcium, and the addition of specific chelators does not affect fungal growth. Hydrophobic derivatives of the calcium-specific chelator BAPTA designed to traverse plant cuticles were synthesized in order to chelate calcium internally during fungal attack. Some chelators as well as calcium precipitating oxalate and channel blocker verapamil were applied with a weakly mycoherbicidal Colletotrichum coccodes to cotyledons of compatible Abutilon threophrasti. They suppressed calcium dependent callose biosynthesis in the weed and increased virulence but may have affected other calcium-dependent processes that facilitate virulence. The low calcium requirement of fungi, and their high affinity for calcium, allows the application of calcium-regulating agents as synergists for mycoherbicides where the weed uses calcium-dependent defenses.

Sixteen convolvulaceous glycolipids selected from the tricolorin (1 - 7) and orizabin (8 - 16) series, proved to be strong in vitro inhibitors of the enzyme that catalyzes the synthesis of 1,3-beta-D-glucan, a major polymer of fungal cell-walls. Results provide an insight into function of the specific structures of these complex macrocyclic lactones as inhibitors of the 1,3-beta-D-glucan synthase and open the possibility of using these compounds as starting points for the development of antifungal agents that act by inhibiting fungal cell-wall synthesis.

In the course of screening for antifungal antibiotics, we have discovered a novel series of lipopeptide compounds structurally related to, but highly superior to, echinocandin B in terms of their water solubility due to the presence of a sulfate residue. These compounds, WF11899s, WF738s, WF14573s, WF16616 and WF22210, and their derivatives have diversity in their nuclear structures and acyl side chains. The producing strains were classified into two groups, the Coleomycetes group and the Hyphomycetes group. Compound FK463, a derivative of WF11899A, is currently in Phase 3 clinical development as a novel antifungal antibiotic.

1,3-beta-glucan synthase, a multisubunit enzyme, is responsible for fungal cell wall construction, division septum deposition, and ascospore wall assembly. The catalytic subunit of this enzyme complex, an integral membrane protein, has been identified both in model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and in pathogenic fungi such as Candida, Aspergillus, Cryptococcus and Pneumocystis species. The catalytic activity of the 1,3-beta-glucan synthase is regulated by a small GTPase of the Ras superfamily, the Rho-GTPase, and protein kinase C (Pkc)-like signaling molecules. It has been shown that the plasma membrane localization of this enzyme is essential for its activity. Interestingly, inhibition of 1,3-beta-glucan synthase activity by anti-fungal drugs of the lipopeptide type triggers a cell cycle feedback mechanism leading to cell cycle arrest. Recent progress in studies of molecular mechanisms of the temporal and spatial regulation of 1,3-beta-glucan synthase is presented. The implication of the cell cycle checkpoint that is activated by the anti-fungal drugs is also discussed.

1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.

Echinocandins, the lipopeptide class of glucan synthase inhibitors, are an alternative to ergosterol-synthesis inhibitors to treat candidiasis and aspergillosis. Their oral absorption, however, is low and they can only be used parenterally. During a natural product screening program for novel types of glucan synthesis inhibitors with improved bioavailability, a fungal extract was found that inhibited the growth of both a wild-type Saccharomyces cerevisiae strain and the null mutant of the FKS1 gene (fks1::HIS). The mutant strain was more sensitive to growth inhibition, suggesting that the fungal extract could contain an inhibitor of glucan synthesis. A novel acidic steroid, named arundifungin, was purified from a fungal extract obtained from a liquid culture of Arthrinium arundinis collected in Costa Rica. Arundifungin caused the same pattern of hallmark morphological alterations in Aspergillus fumigatus hyphae as echinocandins, further supporting the idea that arundifungin belongs to a new class of glucan synthesis inhibitors. Moreover, its antifungal spectrum was comparable to those of echinocandins and papulacandins, preferentially inhibiting the growth of Candida and Aspergillus strains, with very poor activity against Cryptococcus. Arundifungin was also detected in nine other fungal isolates which were ecologically and taxonomically unrelated, as assessed by sequencing of the ITS1 region. Further, it was also found in two more Arthrinium spp from tropical and temperate regions, in five psychrotolerant conspecific isolates collected on Macquarie Island (South Pacific) and belonging to the Leotiales, and in two endophytes collected in central Spain (a sterile fungus belonging to the Leotiales and an undetermined coelomycete).

Highly potent 1,3-beta-D-glucan synthase inhibitors, 7b, 10a, 10b and 12, have been identified by the chemical modification of the ornithine residue of a fungicidal macrocyclic lipopeptidolactone, RO-09-3655 (1), isolated from the cultured broth of Deuteromycotinia spp. These compounds showed stronger antifungal activity against systemic candidiasis as well as pulmonary aspergillosis in mice, and less hepatotoxicity as compared with 1.

The synthesis and biological properties of a novel water-soluble echinocandin-like lipopeptide, FR131535, are described. This compound displayed potent in vitro and in vivo antifungal activities. The hemolytic activity of FR901379 was reduced by replacing the acyl side chain. This compound showed good water-solubility, comparable to the natural product FR901379.

Highly potent 1,3-beta-D-glucan synthase inhibitors 10, 11 and 13 have been identified by the chemical modification of the fungicidal macrocyclic lipopeptidolactone, RO-09-3655 (1), isolated from the cultured broth of Deuteromycotinia spp. D-Ornithine derivative (10) showed improved antifungal activity in the systemic candidiasis model in mice and reduced hepatotoxicity in vitro, as compared with 1.

Extracts of the biomasses and fermentation broths of 217 extremophilic microorganisms isolated from a number of locales were screened for antifungal activity using whole-cell and mechanism-based in vitro assays. Importantly, eleven broth extracts had activity against several Candida species and Aspergillus fumigatus in whole-cell in vitro assays. One broth specifically inhibited (1,3)beta-glucan synthase activity and four specifically inhibited ketol-isomerase activity, suggesting a mode of action of the antifungal compound(s) present in these extracts. The extract from one thermophile, a novel species of Pseudomonas, was fractionated, an active compound purified and its structure determined. The compound was identified as pyochelin, a previously identified iron-binding compound with heretofore undescribed antifungal activity. To our knowledge, this is the first report demonstrating that extremophiles synthesize compounds that have antifungal activity.

Arborcandins A, B, C, D, E and F, which possess potent 1,3-beta-glucan synthase inhibitory activity, were isolated from the culture broth of a filamentous fungus, strain SANK 17397. Arborcandins are novel cyclic peptides, that are structurally different from known glucan synthase inhibitors such as echinocandins. The 1,3-beta-glucan synthases of Candida albicans and Aspergillus fumigatus were inhibited by arborcandins with IC50 ranging from 0.012 to 3 microg/ml. The apparent Ki value of arborcandin C for C. albicans and A. fumigatus were 0.12 microM and 0.016 microM, respectively. The inhibition against these two 1,3-beta-glucan synthases by arborcandin C was noncompetitive. These compounds exhibited potent fungicidal activity against Candida spp. with MIC ranging from 0.25 to 8 microg/ml. The growth of A. fumigatus was suppressed by arborcandins with concentrations ranging from 0.063 to 4 microg/ml.

This study examined the inhibitory effects of trans-cinnamaldehyde (CA), an aromatic aldehyde derived from Cinnamomi Cortex, on Saccharomyces cerevisiae cell wall synthesizing enzymes in vitro. This compound was found to be a noncompetitive inhibitor of beta-(1,3)-glucan synthase and a mixed inhibitor of chitin synthase 1 with 50% inhibitory concentrations (IC50) of 0.84 and 1.44 mM, respectively. Chitin synthases 2 and 3 were less sensitive than chitin synthase 1 to CA. CA can be useful as a model compound of cell wall inhibitors for the development of effective antifungal agents.

The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Natural-product inhibitors of cell wall (1,3)-beta-D-glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades. As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin B(o), is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis. This and other lipopeptide antifungal agents have limited oral bioavailability. Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption. Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-beta-D-glucan synthase inhibitors. Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991. Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-beta-D-glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in Saccharomyces cerevisiae strains with point mutations in FKS1, the gene which encodes the large subunit of glucan synthase.

We have isolated a new gene, psu1(+), from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequences shows that this protein has striking homology to the SUN family of the budding yeast, hence designated Psu1 (S. pombe homologue of the SUN family). Disruption of the psu1(+) gene revealed that it is essential for growth, and the null phenotype showed the swelling of cells followed by eventual lysis. We introduced psu1(+) gene in the disruptant strain and repressed it giving resistance to 1, 3-beta-glucanase digestion. Our results suggest that Psu1 plays an essential role in cell wall synthesis in S. pombe., (Copyright 1999 Academic Press.)

Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1(E45I) when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I). Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.

We investigated the efficacy of non-azole antifungal agents. Long circulating immunoliposomal amphotericin B was potent in murine invasive pulmonary aspergillosis. The concentration of AMPH-B was still high in the lung after 6 hours of 34A-PEG-liposomal AMPH-B. Lipid nanosphere amphotericin B (NS-718) showed efficacy against pulmonary aspergillosis in rats and pulmonary cryptococcosis in mice. The renal toxicity of NS-718 was estimated to be lower than that of AMPH-B from the results of the toxicity study in the rat infusion model. FK 463, a novel (1,3)-beta-D-glucan synthase inhibitor, showed efficacy against azole-resistant Candida albicans in murine experimental disseminated candidiasis. FK463 could be a promising drug and the therapy of choice for azole resistant C. albicans infection.

The mechanism of the killing and cytocidal effects produced by HYI toxin from Williopsis saturnus var. saturnus, and by the amphiphilic antibiotics aculeacin A and papulacandin B on yeast Saccharomyces bayanus cells was studied. When the yeast cells were treated with these molecules, a discharge of cell materials at the budding position was observed by phase-contrast and scanning electron microscopy. The cytocidal effect of these molecules was most pronounced when the cells were in the logarithmic growth phase. Washing the HYI toxin incubation mixture completely eliminated the killing activity, but in the case of the antibiotics, it only partially reduced the cytocidal activity. Full recovery of the killing activity in the supernatant of the washing solution was observed after HYI toxin incubation, but in the case of the antibiotics, the recovery of cytocidal activity was time-dependent. The activity of membrane beta-1,3-glucan synthase was potently inhibited by HYI toxin, and the concentration of this enzyme in the budding tip was observed. These results suggest that HYI toxin exerts a cytocidal effect on the budding of sensitive yeast cells by inhibiting cell wall synthesis. This mechanism is similar to that of the HM-1 toxin of W. saturnus var. mrakii, and to aculeacin A and papulacandin B, although there are some differences from that of HYI toxin.

(1,3)Beta-D-glucan synthase (E.C.2.4.1.34. UDP-glucose: 1,3-beta-D-glucan 3-beta-glucosyl transferase) catalyzes the polymerization of glucose ([1-3]-beta-linkages) using UDP-glucose as substrate. We have determined optimal in vitro conditions for the assay of (1,3)beta-glucan synthase activity from Aspergillus fumigatus and Candida albicans. These included lysis of cells in the following for C. albicans, 100 mM HEPES, pH 8.0, 10 microM guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), 2 mM ethylenediaminetetraacetic acid (EDTA), disodium salt, 5 mM NaF, 250 mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50 mM HEPES, 10mM EDTA, 750 mM sucrose, 10 mM NaH2PO4, 100 mM cellobiose and 50 microM GTPgammaS. Resulting low-speed supernatants were used as enzyme sources to determine the optimal in vitro assay conditions. We have characterized the resulting enzyme activities and tested the optimized assays with known (1,3)beta-glucan synthase inhibitors including cilofungin, papulacandin, aculeacin A, and echinocandin B. We have used both optimized assays to screen > 1000 extracts of marine macroorganisms and, using bioassay-guided purification, have identified (1,3)beta-glucan synthase inhibitors.

Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

In pollen tubes of Nicotiana alata, a membrane-bound, Ca(2+)-independent callose synthase (CalS) is responsible for the biosynthesis of the (1,3)-beta-glucan backbone of callose, the main cell wall component. Digitonin increases CalS activity 3- to 4-fold over a wide range of concentrations, increasing the maximum initial velocity without altering the Michaelis constant for UDP-glucose. The CalS activity that requires digitonin for assay (the latent CalS activity) is not inhibited by the membrane-impermeant, active site-directed reagent UDP-pyridoxal when the reaction is conducted in the absence of digitonin. This is consistent with digitonin increasing CalS activity by the permeabilization of membrane vesicles. A second group of detergents, including 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Zwittergent 3-16, and 1-alpha-lysolecithin, activate pollen tube CalS 10- to 15-fold, but only over a narrow range of concentrations just below their respective critical micellar concentrations. This activation could not be attributed to any particular chemical feature of these detergents. CHAPS increases maximum initial velocity and decreases the Michaelis constant for UDP-glucose and activates CalS even in the presence of permeabilizing concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate that activation by CHAPS occurs by recruitment of previously inactive CalS molecules to the pool of active enzyme. The activation of pollen tube CalS by these detergents therefore resembles activation of the enzyme by trypsin.

We have used the filamentous fungus, Neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)beta-glucan synthase [E.C. 2.4.1.34; UDPglucose: 1,3-beta-D-glucan 3-beta-D-glucosyltransferase], leads to a corresponding decrease in growth of the organism. Previously, our laboratory isolated a gene (glucan synthase-1, gs-1) that is required for (1,3)beta-glucan synthase activity. Wild-type cells were transformed with DNA vectors encoding various RNAs complementary to the gs-1 messenger RNA (antisense RNA) cloned downstream from an inducible promoter (quinic acid-2[qa-2p]). Stable transformants, expressing a partially inverted antisense message of gs-1 (pMYX107), exhibited dramatic reduction ingrowth compared with empty vector controls. Hyphal measurements of these transformants grown on race tubes indicated that all of the transformants showed various degrees of inhibition. Microscopic observations of transformants revealed shorter hyphal lengths when grown under conditions expressing antisense. Further characterization revealed that the specific activities of (1,3)beta-glucan synthase were decreased by as much as 63% relative to empty vector controls. Together, these observations suggest that antisense against (1,3)beta-glucan synthase led to a reduction in enzyme levels that resulted in altered cell-wall morphology and inhibition of growth. It is possible that antisense oligonucleotides against gs-1 may be useful antifungal agents.

The echinocandins and pneumocandins are lipopeptide antifungal agents that inhibit the synthesis of 1,3-beta-D-glucan, an essential cell wall homopolysaccharide found in many pathogenic fungi. Compounds with this fungal-specific target have several attractive features: lack of mechanism-based toxicity, potential for fungicidal activity and activity against strains with intrinsic or acquired resistance mechanisms for existing antimycotics. Semi-synthetic analogues of naturally occurring lipopeptides are currently in clinical trials with the aim of treating systemic candidiasis and aspergillosis. Thus a fuller understanding of the target enzyme and its inhibition by these compounds should be useful for epidemiological and other clinical studies. Although it has been long known that lipopeptides inhibit fungal glucan synthase activity both in cell extracts and in whole cells, the genetic and biochemical identification of the proteins involved has been accomplished only recently. We now know that in Saccharomyces cerevisiae, glucan synthase is a heteromeric enzyme complex comprising one large integral membrane protein (specified by either FKS1 or by FKS2) and one small subunit more loosely associated with the membrane (specified by RHO1). Additional components may also be involved. The heteromeric enzyme complex containing Fks1p constitutes the majority of the activity found in vegetatively growing cells in this organism. The FKS2 gene product is needed for sporulation. Lipopeptides affect the function of the Fksp component from either FKS gene. The current model for interaction and regulation of these components in S. cerevisiae and the application to Candida albicans and other pathogenic fungi are discussed in this review.

We have investigated the molecular basis for the reported synergism between peptaibols and cell wall hydrolytic enzymes in the antagonism of phytopathogenic fungi by Trichoderma harzianum. beta-Glucan synthase activity on isolated plasma membranes of Botrytis cinerea was inhibited in vitro by the peptaibols trichorzianin TA and TB, and this inhibition was reversed by the addition of phosphatidylcholine. beta-Glucan synthesis in vivo, assayed by the incorporation of [2-(3)H]glucose into cell wall material, was inhibited by the presence of peptaibols, and this inhibition was synergistic with exogenously added T. harzianum beta-1,3-glucanase. This synergism is therefore explained by an inhibition of the membrane-bound beta-1,3-glucan synthase of the host by the peptaibols, which inhibit the resynthesis of cell wall beta-glucans, sustain the disruptive action of beta-glucanases, and all together enhance the fungicidal activity. Therefore, we have identified cell wall turnover as a major target of mycoparasitic antagonism.

The incidence of severe, life-threatening fungal infections has increased dramatically over the last decade. Unfortunately, in practice the arsenal of antifungal drugs is limited to flucytosine, a few approved azoles, and polyenes, mainly amphotericin B. This situation is rather precarious in view of the extended spectrum of fungi causing severe disease in immunocompromised patients, development of resistance to some of the currently used agents, and the minimal fungicidal activity of the azoles. Although lagging behind the need for new antifungal agents, the study of fungal biochemistry, physiology, and genetics has undergone a resurgence to new heights of activity, thus providing a framework on which to build drug discovery programs in several new areas, two of which will be discussed in detail: the biology of Candida albicans secreted aspartyl protease with respect to inhibitor discovery, evaluation, and possible clinical utility; and the fungal cell wall beta-glucans with respect to the mechanism and regulation of synthesis and target sites for drug inhibition.

Papulacandin B, an antifungal agent that interferes with the synthesis of yeast cell wall (1,3)beta-D-glucan, was used to isolate resistant mutants in Schizosaccharomyces pombe and Saccharomyces cerevisiae. The resistance to papulacandin B always segregated as a recessive character that defines a single complementation group in both yeasts (pbr1+ and PBR1, respectively). Determination of several kinetic parameters of (1,3)beta-D-glucan synthase activity revealed no differences between S. pombe wild-type and pbr1 mutant strains except in the 50% inhibitory concentration for papulacandin B of the synthases (about a 50-fold increase in mutant activity). Inactivation of the synthase activity of both yeasts after in vivo treatment with the antifungal agent showed that mutant synthases were more resistant than the corresponding wild-type ones. Detergent dissociation of the S. pombe synthase into soluble and particulate fractions and subsequent reconstitution indicated that the resistance character of pbr1 mutants resides in the particulate fraction of the enzyme. Cloning and sequencing of PBR1 from S. cerevisiae revealed a gene identical to others recently reported (FKS1, ETG1, CWH53, and CND1). Its disruption leads to reduced levels of both (1,3)beta-D-glucan synthase activity and the alkali-insoluble cell wall fraction. Transformants containing the PBR1 gene reverse the defect in (1,3)beta-D-glucan synthase. It is concluded that Pbr1p is probably part of the (1,3)beta-D-glucan synthase complex.

1,3-beta-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p. The residual glucan synthase activity present in strains with deletions of fks1 is (i) immunodepleted by antibodies prepared against FKS2 peptides, demonstrating that Fks2p is also a component of the enzyme, and (ii) more sensitive to the echinocandin L-733,560, explaining the increased sensitivity of fks1 null mutants to this drug. Simultaneous disruption of FKS1 and FKS2 is lethal, suggesting that Fks1p and Fks2p are alternative subunits with essential overlapping function. Analysis of FKS1 and FKS2 expression reveals that transcription of FKS1 is regulated in the cell cycle and predominates during growth on glucose, while FKS2 is expressed in the absence of glucose. FKS2 is essential for sporulation, a process which occurs during nutritional starvation. FKS2 is induced by the addition of Ca2+ to the growth medium, and this induction is completely dependent on the Ca2+/calmodulin-dependent phosphoprotein phosphatase calcineurin. We have previously shown that growth of fks1 null mutants is highly sensitive to the calcineurin inhibitors FK506 and cyclosporin A. Expression of FKS2 from the heterologous ADH1 promoter results in FK506-resistant growth. Thus, the sensitivity of fks1 mutants to these drugs can be explained by the calcineurin-dependent transcription of FKS2. Moreover, FKS2 is also highly induced in response to pheromone in a calcineurin-dependent manner, suggesting that FKS2 may also play a role in the remodeling of the cell wall during the mating process.

Glucan synthesis was sensitive to several sulfhydryl reacting compounds: mercurials, reversible disulfides, and an alkylating sulfhydryl reagent (IC50 3-45 microM). Thiol groups associated with glucan synthesis were hydrophilic in nature, since both hydrophilic and hydrophobic reagents were active. Glucan synthase complex consists of at least two components: a peripheral GTP-binding protein that can be solubilized with detergents (supernatant) and the catalytic membrane-bound component (pellet). A rapid separation technique was developed to study sulfhydryl interactions with the complex. The GTP-binding protein was solubilized with 0.6% 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate from isolated microsomes of Candida albicans cells grown at either 10 or 30 degrees C. The residual membranous fraction contained the core catalytic moiety of glucan synthase. Both fractions were devoid of glucan synthase activity until they were reconstituted by mixing the two fractions together. In reconstitution experiments, the pellet lost almost 50% activity when preincubated with 2.5 microM N-ethylmaleimide and combined with an untreated supernatant whereas only 10% activity was lost when the supernatant was treated with N-ethylmaleimide. The catalytic active site of glucan synthase was not protected with UDP-Glc when preincubated with 10 microM N-ethylmaleimide but the GTP-binding fraction was partially protected with GTP gamma S.

HM-1 toxin produced by Hansenula mrakii kills sensitive Saccharomyces cerevisiae. We found that the budding cells and the cells that responded to mating factor were sensitive to HM-1 toxin. These findings indicate that the target sites of HM-1 toxin are developing buds and conjugating tubes. The in vitro activity of beta-1,3-glucan synthase solubilized and partially purified from S. cerevisiae membranes was inhibited by HM-1 toxin at a concentration (around 50 nM, 0.5 micrograms/ml) that coincided well with its minimum inhibitory concentration for the growth of yeast cells. These data indicate that the HM-1 toxin perturbs the synthesis of yeast cell walls by inhibiting the glucan synthesis occurring at a budding site or a conjugating tube, which results in cell lysis.

WF11899A, B and C, novel water-soluble lipopeptides related to the echinocandins, possess potent anti-Candida activities. The IC50s of the compounds against four clinical isolates of Candida albicans ranged from 0.004 to 0.03 microgram/ml by microbroth dilution assay. These compounds mildly suppressed the growth of Aspergillus fumigatus and A. niger. WF11899A, B and C showed a potent in vivo anti-Candida activity. Particularly, WF11899A was superior to cilofungin, and equal to fluconazole. 1,3-beta-glucan synthase was inhibited by these compounds at the IC50s of 0.7, 0.7 and 1.8 micrograms/ml for WF11899A, B and C, respectively. However, they hemolysed mouse red blood cells in vitro at the concentration of 62 micrograms/ml.

A novel, potent, semisynthetic pneumocandin, L-733,560, was used to isolate a resistant mutant in Saccharomyces cerevisiae. This compound, like other pneumocandins and echinocandins, inhibits 1,3-beta-D-glucan synthase from Candida albicans (F.A. Bouffard, R.A. Zambias, J. F. Dropinski, J.M. Balkovec, M.L. Hammond, G.K. Abruzzo, K.F. Bartizal, J.A. Marrinan, M. B. Kurtz, D.C. McFadden, K.H. Nollstadt, M.A. Powles, and D.M. Schmatz, J. Med. Chem. 37:222-225, 1994). Glucan synthesis catalyzed by a crude membrane fraction prepared from the S. cerevisiae mutant R560-1C was resistant to inhibition by L-733,560. The nearly 50-fold increase in the 50% inhibitory concentration against glucan synthase was commensurate with the increase in whole-cell resistance. R560-1C was cross-resistant to other inhibitors of C. albicans 1,3-beta-D-glucan synthase (aculeacin A, dihydropapulacandin, and others) but not to compounds with different modes of action. Genetic analysis revealed that enzyme and whole-cell pneumocandin resistance was due to a single mutant gene, designated etg1-1 (echinocandin target gene 1), which was semidominant in heterozygous diploids. The etg1-1 mutation did not confer enhanced ability to metabolize L-733,560 and had no effect on the membrane-bound enzymes chitin synthase I and squalene synthase. Alkali-soluble beta-glucan synthesized by crude microsomes from R560-1C was indistinguishable from the wild-type product. 1,3-beta-D-Glucan synthase activity from R560-1C was fractionated with NaCl and Tergitol NP-40; reconstitution with fractions from wild-type membranes revealed that drug resistance is associated with the insoluble membrane fraction. We propose that the etg1-1 mutant gene encodes a subunit of the 1,3-beta-D-glucan synthase complex.

An in vitro assay for (1,3)beta-glucan synthase activity from the filamentous ascomycete Neurospora crassa, suitable for use as a high throughput screen for enzyme inhibitors is described. Samples were added to 25 microliters reaction mixtures in 96-well V-bottom microtiter plates and plates incubated at 22 degrees C. Reactions were terminated by the addition of TCA and the contents transferred to a Milliblot D apparatus containing Inotech 201-A glass fiber filters. Filters were washed to remove unincorporated substrate and the amount of 14C-labeled (1,3)beta-glucan formed by each reaction was quantitated using a Phosphorlmager. As little as 50 ng of an inhibitor with a Ki of 4 microM was detected by this assay. This assay is rapid and cost effective, permitting its use as a screen to detect compounds that inhibit (1,3)beta-glucan synthase activity.

A systematic evaluation of the in vitro (1,3)-beta-glucan synthase assay parameters was performed using microsomes prepared from Candida albicans from either yeast or mycelial phase cells. Enzyme activities of both yeast and mycelial phase microsomes depended on the presence of guanosine-5'-O-(3-thiophosphate) and either bovine serum albumin or a detergent [W-1 (polyoxyethylene ether detergent) or Brij-35 (polyoxyethylene ether, 23 lauryl ether)]. Brij-35 was included in standard assays as it was compatible with the permeabilized whole-cell assay. Microsomes derived from both the yeast and mycelial phases generally yielded similar glucan synthase activities under a range of different assay conditions. Brij-35 significantly stabilized the enzyme, yielding a half-life of 5.6 d at 4 degrees C, compared with 0.9 d without detergent. The addition of detergent during mechanical breakage of yeast cells dramatically improved glucan synthase stability and activity. Enzyme catalysis was linear for at least 75 min with 100 micrograms protein from microsomes of yeast cells grown to mid-exponential phase, with an apparent Km for UDP-glucose of 1.1 mM. The pH and temperature optima were 7.75 and 30 degrees C, respectively. Glucan synthase activity was highest in cells derived from early mid-exponential phase and declined to a basal level by stationary phase. A permeabilization-based in situ assay for glucan synthase was developed. Cells were permeabilized with 2% (v/v) solution of toluene/methanol (1:1) and assayed for glucan synthase activity using standard reaction mixtures. Reactions were linear for 30 min and were inhibited by known inhibitors of glucan synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Fungal (1,3)-beta-glucan synthases are sensitive to a wide range of lipophilic inhibitors and it has been proposed that enzyme activity is highly sensitive to perturbations of the membrane environment. Yeast membranes were exposed to phospholipases and various lipophilic compounds, and the resultant effects on glucan synthase activity were ascertained. Glucan synthase from Saccharomyces cerevisiae was rapidly inactivated by phospholipase A2 (PLA2), and to a lesser extent by phospholipase C. Inactivation was time and dose-dependent and was protected against by EDTA and fatty-acid binding proteins (bovine and human serum albumins). Albumins also partially protected against inhibition by papulacandin B. PLA2 reaction products were structurally characterized and it was shown that fatty acids and lysophospholipids were the inhibitory moieties, with no novel inhibitory compounds apparent. Glucan synthase was inhibited by a range of fatty acids, monoglycerides and lysophospholipids. Inhibition by fatty acids was non-competitive, and progressive binding of [14C]oleic acid correlated with activity loss. Fluorescence anisotropy studies using diphenylhexatriene (DPH) confirm that fatty acids increase membrane fluidity. These results are consistent with proposals suggesting that glucan synthase inhibition is due in part to non-specific detergent-like disruption of the membrane environment, in addition to direct interactions of lipophilic inhibitors with specific target sites on the enzyme complex.

The lipopeptide antifungal agents, echinocandins, papulacandins, and pneumocandins, kill Candida albicans by inhibiting glucan synthesis. For this fungus, there is a good correlation of in vitro enzyme inhibition with in vitro assays of MICs. Semisynthetic lipopeptides such as cilofungin, LY303366, L-693,989, and L-733,560 have activity in vivo against Aspergillus infections but appear to be inactive in broth dilution in vitro tests (MICs, > 128 micrograms/ml). To understand how compounds which lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology of Aspergillus fumigatus and A, flavus strains by light microscopy and electron microscopy and related the changes in growth to inhibition of glucan synthesis. Pneumocandin B0 caused profound changes in hyphal growth; light micrographs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron micrographs confirmed that lipopeptides produce changes in cell walls; drug-treated germlings showed very stubby growth with thick walls and a conspicuous dark outer layer which was much thicker in the subapical regions. The rest of the hyphal tip ultrastructure was unaffected by the drug, indicating considerable specificity for the primary target. The drug-induced growth alteration produced very compact clumps in broth dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitatively by using conventional broth microdilution susceptibility assay conditions. We defined the endpoint as the lowest concentration required to produce the morphological effect and called it the minimum effective concentration to distinguish it from the no-growth endpoints used in MIC determinations. The minimum effective concentration assay was related to inhibition of glucan synthase activity in vitro and may provide a starting point for development of susceptibility testing methods for lipopeptides.

(1,3)-beta-D-Glucan synthase, a major cell wall synthesis enzyme, is the target of antifungal drugs of the lipopeptide class. Aspergillus fumigatus (1,3)-beta-D-glucan synthase was prepared and its activity was measured by incorporation of [14C]glucose from UDP-[U-14C]glucose into an insoluble polymer in the presence of alpha-amylase. Solubilization of the (1,3)-beta-D-glucan synthase was attempted with several detergents, and the maximum percent solubilization was obtained with a polyoxyethylene ether detergent, W-1. Up to 70% of enzyme activity and 50% of total protein were recovered when 1-mg/ml membrane preparations were extracted with 0.045% W-1 at 4 degrees C overnight. Confirmation of the presence of a (1,3)-beta-D-glucose polymer synthesized by this glucan synthase was done by three methods. The first was enzymatic end product degradation by alpha-amylase (no degradation) and beta-glucanase (85 to 95% degradation). The second was gas chromatography-mass spectroscopy analysis of the partially methylated alditol acetate derivatives prepared from total carbohydrate polymers present in the sample. This method identified the presence of (1,3)- and (1,2)-glucosidic linkages. The third was high-performance anion exchange chromatography of radioactive oligosaccharides. This method allowed differentiation of the newly synthesized, radioactive polymers from the contaminating carbohydrates already present in the preparation. The results showed that the polymer synthesized comprised oligosaccharides consistent with beta-(1,3)-linked sugars. Maximal inhibition of the (1,3)-beta-D-glucan synthase by the lipopeptide antifungal agent cilofungin was 80%. Dose-response experiments with this inhibitor showed that the solubilized enzyme was maximally inhibited at a cilofungin concentration of 1.25 microgram/ml and showed <5% inhibition at 0.02 microgram/ml. The apparent K(m) (K(m app)) for the solubilized glucan synthase was 400 +/- 80 microM, and the apparent K(i) (K(i app)) for cilofungin was 0.19 +/- 0.03 microM. Inhibition of A.fumigatus (1,3)-beta-D-glucan synthase with cilofungin was noncompetitive, as it was for the Candida albicans (1,3)-beta-D-glucan synthase.

Rylux BSU, a new fluorescent brightener from the family of 4,4'-diaminostilbene-2,2'disulfonic acid derivatives, inhibited growth and cytokinesis of the yeast Saccharomyces cerevisiae. In the presence of 0.1-1 mg/ml Rylux BSU the cells grew in clumps, had irregular shape and were larger than controls. They formed apparently normal primary septa but their secondary septa and lateral cell walls, especially those in older cells, were abnormally thick with large deposits of amorphous wall material in the periplasmic spaces all over the cell surface. Chitin content in the cell walls of cells grown in the presence of Rylux BSU was increased 2 to 5 times in comparison to that of the controls and glucan content was reduced by up to 30%. In the in vitro assays with particulate membrane fractions, Rylux BSU acted as a non-competitive inhibitor of beta-1,3-glucan synthase with inhibitory constant Ki = 1.75 mg/ml whereas the chitin synthase was inhibited to a much lesser extent. From the difference of the effects of Rylux BSU on the synthesis of chitin in vivo and in vitro it is concluded that the brightener interacts with chitin synthase only indirectly, possibly by influencing the properties of integral plasma membrane.

1,3-beta-Glucan synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors--GTP, NaF, sucrose and EDTA--added during the extraction procedure, were essential for optimal 1,3-beta-glucan synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a Ki of 1.42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (Km for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.

New beta-1,3-glucan synthase inhibitor (BU-4794F) was isolated from the culture broth of Gilmaniella sp. FA4459. Structural studies indicated that it was a novel member of the papulacandin group of antibiotics.

Cotton fibers contain a characteristic set of proteins which interact with plasma membranes in a Ca(2+)-dependent manner. The association of these proteins with the membrane is correlated with a reduced level of UDP-glucose: (1-->3)-beta-glucan (callose) synthase activity. Analysis of the proteins released from membranes by EDTA treatment shows that the most abundant proteins comprise a family of at least three polypeptides (p34) which resemble annexins. This resemblance includes similarity in size (about 34 kDa), sequence homology, Ca(2+)-dependent precipitation or interaction with the plasma membrane, and ability to serve as a substrate for phosphorylation by endogenous protein kinase(s) which also bind to the membranes in a Ca(2+)-dependent manner. A purified fraction of these annexins binds to, and inhibits, the activity of a partially purified cotton fiber callose synthase. These findings suggest that one possible function of annexin(s) in plants is to modulate the activity and/or localization of callose synthase.

(1,3)-beta-D-Glucan synthase of Candida albicans was rendered soluble by treatment of membrane preparations with the polyoxyethylene ether detergent W-1. Extraction with 0.025% W-1 at 4 degrees C for 24 h effectively solubilized and activated the enzyme. Under these conditions, greater than 85% of the protein in membrane preparations was released, and about 64% of the glucan synthase activity could be recovered in the soluble form. Soluble enzyme activity was stable for more than 12 days at 4 degrees C. Also, glucan synthase activity in the extracted membrane preparations could be activated to achieve more than twice the enzyme activity in the original, unextracted membrane preparations. The soluble glucan synthase had characteristics similar to those of the membrane-bound enzyme. Soluble glucan synthase had an apparent Km of 2.0 mM, and particulate glucan synthase had an apparent Km of 2.5 mM. Kinetics of cilofungin inhibition for both enzyme preparations were noncompetitive, with an apparent Ki of 2.5 microM; both preparations could be inhibited by cilofungin but not by its peptide nucleus or side chain, either alone or in combination. The reaction products from both forms of the enzyme were sensitive to (1,3)-beta-D-glucanase degradation but not to alpha-amylase, alpha-glucosidase, or proteinase K degradation and thus were shown to be beta(1----3) glucan.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.