Effect of allylamine antimycotic agents on fungal sterol biosynthesis measured by sterol side-chain methylation

Sterol side-chain (C-24) methylation was assayed by incorporation of radioactivity from [Me-14C]methionine into the ergosterol fraction in cells of the pathogenic fungi Candida albicans, Candida parapsilosis and Trichophyton mentagrophytes. Methylation at C-24 occurred after nuclear demethylation in all cases. The method was used to measure ergosterol biosynthesis inhibition by the allylamine antimycotics naftifine and SF 86-327, which are known to block squalene epoxidation. In C. albicans cells treated with SF 86-327 (1 mg l-1) to fully inhibit squalene epoxidation, C-24 methylation continued for several hours at about 40% of the control rate. This residual biosynthesis was probably due to methylation of endogenous sterol precursors. The degree of residual biosynthesis in the three fungi correlated well with their susceptibility to SF 86-327. The highly susceptible dermatophyte T. mentagrophytes had negligible residual sterol biosynthesis. These differences were not due to inhibition of methionine uptake. For naftifine (100 mg l-1) there was evidence of a second inhibitory action in C. albicans. A cell-free assay indicated that this was due to direct inhibition of the C-24 methyltransferase.     

In-vitro studies with SF 86-327, a new orally active allylamine derivative

SF 86-327 (Sandoz Forschungsinstitut) is an orally active allylamine derivative related to naftifine. The antifungal activity of SF 86-327 was compared in vitro with those of naftifine, ketoconazole, and itraconazole (R 51,211, Janssen Pharmaceutica) by agar dilution. 120 fungal isolates were tested. Also, the antifungal activities of SF 86-327 and naftifine against 18 dimorphic pathogens were assayed in vitro by broth dilution. Results of these studies support claims that SF 86-327 is a broad spectrum antifungal agent. Results of the broth dilution studies also revealed that SF 86-327 was both fungistatic and fungicidal in vitro for isolates of Blastomyces dermatitidis, Histoplasma capsulatum and Sporothrix schenckii at concentrations as low as 0.05 micrograms ml-1 (18 isolates tested, MIC90 = 0.39 micrograms ml-1, MFC90 = 12.5 micrograms ml-1).     

白念珠菌角鲨烯环氧化酶基因开放读框的克隆及其在大肠杆菌中的表达*

根据白念珠菌角鲨烯环氧化酶基因的开放读框中编码1MSSVKY6的序列和编码492NEIVR496的序列分别设计上、下游引物,以白念珠菌ATCC11006的基因组DNA为模板进行PCR扩增;将PCR产物克隆并做序列分析后,在大肠杆菌中进行表达。结果表明PCR获得大小约为1.5kb的产物,测序分析表明克隆的产物大小为1491bp,正是白念珠菌角鲨烯环氧化酶基因的开放读框,表达得到约为80kDa大小的蛋白,与理论计算一致。本研究为开展特比萘芬与其作用靶酶关系的研究奠定了基础。     

Properties of a particulate squalene epoxidase from Candida albicans

The properties and requirements of squalene epoxidase and effects of some inhibitors were investigated in the pathogenic yeast Candida albicans. A washed 'microsomal' fraction converted radiolabelled squalene to 2,3-oxidosqualene and lanosterol. Minimum requirements for activity were molecular oxygen, NADH or NADPH, and FAD. Epoxidase activity was stimulated by up to 100% by addition of the soluble cytoplasmic fraction, which itself contained negligible epoxidase activity. This stimulation was most powerful at low concentrations of enzyme, or high concentrations of squalene. Divalent cations did not stimulate activity and EDTA was not inhibitory. An apparent Km for squalene of 50 microM was determined in the presence of soluble cytoplasm. Epoxidase activity was destroyed by Triton X-100, deoxycholate or Cu2+, and partially inhibited by thiol reagents, rotenone and antimycin A. The enzyme was not inhibited by cyanide or by several inhibitors of cytochrome P-450.     

Evaluation of Candida species' susceptibility to fluconazole with the disk diffusion method

Infections caused by yeasts belonging to the genus Candida have increased dramatically in the last decades, especially in hospital settings. Concomittantly, antimycotic resistance has emerged, as well as the appearance of non-Candida albicans isolates. To standardize in vitro antifungal susceptibility tests, the agar diffusion test was developed using disks impregnated with the antimycotic compound. Electronic recording of the inhibition zone (BIOMIC), furnishes objective values for the minimal inhibitory concentration (MIC). The fluconazole susceptibility patterns were determined for Candida species isolated from 2.139 patients seen in outpatient clinics or in health-care centers in Colombia, Ecuador and Venezuela. Candida albicans was the species most frequently isolated (62%), followed at a distance by Candida parapsilosis (11%), Candida tropicalis (8.5%), Candida glabata (3.5%) and Candida krusei (2.2%). MIC determinations showed that 88.1% of these isolates were susceptible to fluconazole, 5.1% were susceptible-dose-dependant and 6.8% resistant. An important proportion (92.1%) of the C. albicans isolates proved susceptible while resistance predominated in the remaining species. These results indicate that the BIOMIC method is rapid and simple, constituting a suitable tool for the epidemiologic surveillance of resistance in Candida species.     

Regulation of squalene epoxidase activity and comparison of catalytic properties of rat liver and Chinese hamster ovary cell-derived enzymes

Squalene epoxidase activity has been studied in cell-free preparations of Chinese hamster ovary (CHO) cells and rat liver. In contrast to rat liver microsomal squalene epoxidase, the enzyme of CHO cells is only slightly activated by the autologous cytosolic fraction, whereas phosphatidylglycerol or rat liver cytosolic preparations are potent stimulators of this enzyme. Triton X-100, a known stimulator of the hepatic squalene epoxidase, has no activating effect on the enzyme of CHO cells. The squalene epoxidase activity of both rat liver and CHO cells varies significantly according to the lipid content of the growth medium or diet. The changes in enzyme activity are shown to be entirely due to altered microsomal enzyme per se and not to changes in the activating properties of the soluble fraction. These results further support the proposed regulatory role of squalene epoxidase in cholesterogenesis.     

Regulation of squalene epoxidase in HepG2 cells

Regulation of squalene epoxidase in the cholesterol biosynthetic pathway was studied in a human hepatoma cell line, HepG2 cells. Since the squalene epoxidase activity in cell homogenates was found to be stimulated by the addition of Triton X-100, enzyme activity was determined in the presence of this detergent. Incubation of HepG2 cells for 18 h with L-654,969, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased squalene epoxidase activity dose-dependently. On the other hand, low density lipoprotein (LDL) and 25-hydroxy-cholesterol decreased the enzyme activity. These results demonstrate that squalene epoxidase is regulated by the concentrations of endogenous and exogenous sterols. The affinity of the enzyme for squalene was not changed by treatment with L-654,969. Cytosolic (S105) fractions, prepared from HepG2 cells treated with or without L-654,969, had no effect on microsomal squalene epoxidase activity of HepG2 cells, in contrast to the stimulating effect of S105 fractions from rat liver homogenate. Mevalonate, LDL, and oxysterol treatment abolished the effect of L-654,969. Simultaneous addition of cycloheximide and actinomycin D also prevented enzyme induction in HepG2 cells. From these results, the change in squalene epoxidase activity is thought to be caused by the change in the amount of enzyme protein. It is further suggested that squalene epoxidase activity is suppressed only by sterols, not by nonsterol derivative(s) of mevalonate, in contrast to the regulation of HMG-CoA reductase.     

Purification and partial characterization of squalene epoxidase from rat liver microsomes

Squalene epoxidase (EC 1.14.99.7, squalene 2,3-monooxygenase (epoxidizing) was purified to an apparent homogeneity from rat liver microsomes. The purification was carried out by solubilization of microsomes by Triton X-100, fractionation with ion exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatography. A total purification of 143-fold over the first DEAE-cellulose fraction was achieved. The purified enzyme gave a single major band on SDS-polyacrylamide gel electrophoresis and the Mr was estimated to be 51 000 as a single polypeptide chain. The enzyme showed no distinct absorption spectrum in the visible regions. The squalene epoxidase activity was reconstituted with the purified enzyme, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), FAD, NADPH and molecular oxygen in the presence of Triton X-100. The apparent Michaelis constants for squalene and FAD were 13 microM and 5 microM, respectively. The Vmax was about 186 nmol per mg protein per 30 min for 2,3-oxidosqualene. The enzyme activity was not inhibited by potent inhibitors of cytochrome P-450. It is suggested that squalene epoxidase is distinct from cytochrome P-450 isozymes.     

Oxygen requirements for formation and activity of the squalene epoxidase in Saccharomyces cerevisiae.

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the "mitochondrial" fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the "microsomal" fraction. At 0.03% (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent Km for oxygen of 0.38% (vol/vol) was determined from a crude particulate preparation for the epoxidase.     

Squalene epoxidase as hypocholesterolemic drug target revisited

Therapeutic success of statins has distinctly established inhibition of de novo hepatic cholesterol synthesis as an effective approach to lower plasma LDL-cholesterol, the major risk factor for atherosclerosis and coronary heart disease. Statins inhibit HMG CoA reductase, a rate limiting enzyme which catalyses conversion of HMG CoA to mevalonic acid. However, in this process statins also inhibit the synthesis of several non-sterols e.g. dolichols and ubiquinone, which are implicated in side effects observed with statins. This prompted many major pharmaceutical companies in 1990s to target selective cholesterol synthesis beyond farnesyl pyrophosphate. The enzymes squalene synthetase, squalene epoxidase and oxidosqualene cyclase were identified as potential targets. Though inhibitors of these enzymes have been developed, till date no compound has been reported to have entered clinical trials. We evaluated the literature to understand merits and demerits of pursuing squalene epoxidase as a target for hypocholesterolemic drug development. Squalene epoxidase catalyses the conversion of squalene to 2,3-oxidosqualene. Although it has been extensively exploited for antifungal drug development, it has received little attention as a target for hypocholesterolemic drug design. This enzyme though recognized in the early 1970s was cloned 25 years later. This enzyme is an attractive step for pharmacotherapeutic intervention as it is the secondary rate limiting enzyme and blocking cholesterol synthesis at this step may result in accumulation of only squalene which is known to be stable and non toxic. Synthesis of several potent, orally bioavailable inhibitors of squalene epoxidase has been reported from Yamonuchi, Pierre Fabre and Banyu pharmaceuticals. Preclinical studies with these inhibitors have clearly demonstrated the potential of squalene epoxidase inhibitors as hypocholesterolemic agents. Hypochloesterolemic therapy is intended for prolonged duration and safety is an important determinant in clinical success. Lack of clinical trials, despite demonstrated preclinical efficacy by oral route, prompted us to evaluate safety concerns with squalene epoxidase inhibitors. In dogs, NB-598, a potent competitive squalene epoxidase inhibitor has been reported to exhibit signs of dermatitis like toxicity which has been attributed by some reviewers to accumulation of squalene in skin cells. Tellurium, a non-competitive inhibitor of squalene epoxidase has been associated with neuropathy in weanling rats. On the other hand, increased plasma levels of squalene in animals and humans (such as occurring subsequent to dietary olive oil or squalene administration) are safe and associated with beneficial effect such as chemoprevention and hypocholesterolemic activity. In our view, high circulating levels of squalene epoxidase inhibitor may be responsible for dermatitis and neuropathy. Competitive inhibition and pharmacokinetic profile minimizing circulating plasma levels (e.g. by hepatic sequestration and high first pass metabolism) could be important determinants in circumventing safety concerns of squalene epoxidase inhibitors. Recently, cholesterol-lowering effect of green tea has been attributed to potent squalene epoxidase inhibition, which can be consumed in much higher doses without toxicological effect. These facts strengthen optimism for developing clinically safe squalene epoxidase inhibitors. Put in perspective squalene epoxidase appears to be undervalued target which merits attention for development of better hypocholesterolemic drugs.     

In vitro assay of squalene epoxidase of Saccharomyces cerevisiae

We describe a simple assay for measuring squalene epoxidase specific activity in Saccharomyces cerevisiae cell-free extracts, by using [14C] farnesyl pyrophosphate as substrate. Cofactor requirements for activity are FAD and NADPH or NADH, NADPH being the preferred reduced pyridine nucleotide. Squalene epoxidase activity is localized in microsomal fraction and no supernatant soluble factor is required for maximum activity. Microsomal fraction converted farnesyl pyrophosphate into squalene, squalene 2,3-epoxide and lanosterol, showing that squalene 2,3-epoxide-lanosterol cyclase is also a microsome-bound enzyme. We show also that squalene epoxidase activity is not inhibited by ergosterol or lanosterol, but that enzyme synthesis is induced by oxygen.     

Inactivation and activation of various membranal enzymes of the cholesterol biosynthetic pathway by digitonin

The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of secretory acid proteinases from Candida tropicalis, C. parapsilosis and C. albicans.

Acid proteinases secreted by Candida tropicalis and C. parapsilosis were newly isolated. Their physico-chemical and enzymatic properties of molecular weight, pH stability, isoelectric points, specific activity, and N-terminal amino acid sequences were determined and compared with those of a C. albicans acid proteinase. The two acid proteinases secreted by C. parapsilosis were found to be new enzymes in their molecular weights. The acid proteinases from C. tropicalis and C. parapsilosis showed lower activity at neutral pH, less resistance to neutral and alkaline pH than that from C. albicans, and a half or a third of the specific activity of the C. albicans enzyme. These differences seemed to be associated with the difference of pathogenesis between Candida species. Of the 31 N-terminal amino acids, the enzymes of these three Candida species revealed 12 homologous amino acids.     

Sterol biosynthesis by a prokaryote: first in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.     

Antigenic relationship between Candida parapsilosis and Candida albicans serotype B

We examined the antigenic relationship between Candida parapsilosis and C. albicans serotype B with respect to antigenic factors 13 and 13b, specific for the former species and common to both species, respectively. Acetolysis of C. albicans serotype B cell-wall mannan gave six oligosaccharides. Their chemical structure was determined by 1H-nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and partial acid hydrolysis. The structure of the hexasaccharide derived from C. albicans serotype B mannan was alpha-D-Manp-(1-2)-alpha-D-Manp-(1-3)-alpha-D-Manp-(1- 2)-alpha-D-Manp-(1-2)- alpha-D-Manp-(1-2)-D-Man (M6) which is identical to that from C. parapsilosis mannan. Inhibition of two precipitin reaction systems (anti-C. albicans serotype B serum and anti-C. parapsilosis serum to the respective homologous mannan), by oligosaccharides from homologous and heterologous mannans indicated that M6 from either C. albicans serotype B or C. parapsilosis was the most effective inhibitor. Moreover inhibition of the agglutination reaction between factor serum containing anti-factors 13 and 13b and C. albicans serotype B or C. parapsilosis cells by oligosaccharides from both mannans also indicated that the M6s were the most effective inhibitors. These results suggest that the M6s derived from the two species are identical in their chemical structure, although the structures of the whole mannans of the two species are not identical as demonstrated by gel diffusion precipitation patterns, and that M6s may be involved in the specificities of antigenic factors 13 and 13b. The amount of M6 is larger in C. parapsilosis cell-wall mannan, suggesting that high repeating frequency of M6 fragment may induce the antibody specific for C. parapsilosis.     

A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase.

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.     

Enzymatic differentiation of Candida parapsilosis from other Candida spp. in a membrane filtration test

A previously reported enzyme assay on a membrane filter using 4-methylumbelliferyl (4-MU)-N-acetyl-beta-D-galactosaminide, -phosphate and -pyrophosphate as substrates for the differentiation of four Candida spp. has been extended to Candida parapsilosis. The substrate 4-MU-beta-D-glucoside was hydrolyzed by 28 test strains of this species but to a variable extent by seven other yeasts also. For a full enzymatic differentiation of C. parapsilosis from other medical yeasts, a battery of six reactions was required. Of 71 C. parapsilosis positive clinical samples, 4.2% gave a false negative result due to overgrowth by Candida albicans. The present assay is more rapid than a described spectrofluorometric determination of beta-D-glucosidase in a broth, i.e., 9-11 h versus up to >48 h.     

Mode of action of miconazole on yeasts: inhibition of the mitochondrial ATPase

Miconazole [( 1-[2-(2,4 dichlorophenyl)-2-(2,4 dichlorophenyl)methoxy]ethyl]-1 H-imidazole) completely inhibited growth of Saccharomyces cerevisiae and Candida albicans on glycerol at 10 microM . 50 microM was needed to achieve the same effect during growth on glucose. Miconazole inhibited competitively the mitochondrial ATPase of S. cerevisiae with a Ki of 1 microM. F1 activity of the enzyme was not affected. Mutants resistant to miconazole were isolated. The ATPase of these mutants was resistant to 10 microM miconazole. Higher concentrations of miconazole inhibited the ATPase of the plasma membrane. The inhibition of the S. cerevisiae enzyme was competitive with a Ki of 50 microM. The results point to the mitochondrial ATPase as the primary target of miconazole action at least during growth on non-fermentable carbon sources.     

Solubilization and partial characterization of rat liver squalene epoxidase.

The microsomal enzyme system from rat liver which catalyzes squalene epoxidation requires a supernatant protein and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). It has now been found that these two cytoplasmic components can be replaced by Triton X-100. The same detergent solubilizes the microsomal squalene epoxidase and the resulting supernatant can be separated into two components, A and B, by DEAE-cellulose chromatography. Neither Fraction A nor B alone has significant squalene epoxidase activity but combining the two affords a reconstituted system 5-fold higher in specific epoxidase activity than that of the original microsomes. FAD and Triton X-100 in addition to molecular oxygen and NADPH are required in the reconstituted system. Subjecting Fraction A to a second DEAE-cellulose chromatography does not change its specific activity but lowers NADH-ferricyanide reductase activity and the protoheme content to 1/25 and 1/4, respectively. When Fraction B was chromatographed on Sephadex G-200, the specific epoxidase activity tested in the presence of Fraction A was increased 3-fold. This procedure also raised the specific activity of NADPH-cytochrome c reductase activity in Fraction B 3-fold. The reconstituted epoxidase system is not inhibited by either carbon monoxide, potassium cyanide, or o-phenanthrolien but Tiron at 1 mM was inhibitory (50%). Erythrocuprein has no effect on epoxidation. No evidence has been found for the participation of hemoproteins (P450 or cytochrome b5) in squalene epoxidation. Component B appears to be identical with the flavoprotein NADPH-cytochrome c reductase. Component A may be a flavoprotein with an easily dissociable prosthetic group.