Expression and Characterization of CYP51, the Ancient Sterol 14-demethylase Activity for Cytochromes P450 (CYP), in the White-Rot Fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Phanerochaete chrysosporium was the first fungal genome to exhibit more than one hundred cytochrome P450 (CYP) genes for a fungus within its genome (approximately 150). It can metabolize lignocellulose and a wide range of environmental xenobiotics including many carcinogens and pollutants where cytochromes P450 may be involved. In the present paper we describe the heterologous expression and characterization of an ancestral CYP form, sterol 14alpha-demethylase (CYP51-EC1.14.13.70), from this organism. CYP51 was cloned from a cDNA library and expressed in both Escherichia coli, where it exhibited high affinity for azole antifungals, and Saccharomyces cerevisiae. Proof of function was observed by complementation of a conditional knock-down mutant of yeast CYP51. The CYP51 gene was found to be 1956 bases long and contained 7 exons and 6 introns coding for a polypeptide 550 amino acids long (62 kDa). The CYP51 protein exhibited high affinity (k (d) 0.25-0.45 microM) for azole antifungal compounds.     

Nitrate Esters of Heteroaromatic Compounds as Candida albicans CYP51 Enzyme Inhibitors

Four heteroaromatic compounds bearing nitrate esters were selected using a virtual‐screening procedure as putative sterol 14α‐demethylase (CYP51) Candida albicans inhibitors. Compounds were examined for their inhibition on C. albicans growth and biofilm formation as well as for their toxicity. NMR spectroscopy studies, in silico docking, and molecular dynamics simulations were used to investigate further the selectivity of these compounds to fungal CYP51. All compounds exhibited good antimicrobial properties, indicated with low minimal inhibitory concentrations and ability to inhibit formation of fungal biofilm. Moreover, all of the compounds had the ability to inhibit growth of C. albicans cells. N‐(2‐Nitrooxyethyl)‐1Η‐indole‐2‐carboxamide was the only compound with selectivity on C. albicans CYP51 that did not exhibit cytotoxic effect on cells isolated from liver and should be further investigated for selective application in new leads for the treatment of candidiasis.     

Multiple inhibitory effects of garlic extracts on cholesterol biosynthesis in hepatocytes

Exposure of primary rat hepatocytes and human HepG2 cells to water-soluble garlic extracts resulted in the concentration-dependent inhibition of cholesterol biosynthesis at several different enzymatic steps. At low concentrations, sterol biosynthesis from [¹⁴C]acetate was decreased in rat hepatocytes by 23% with an IC₅₀ (half-maximal inhibition) value of 90μg/mL and in HepG2 cells by 28% with an IC₅₀ value of 35 μg/mL. This inhibition was exerted at the level of hydroxymethylglutaryl-COA reductase (MHG-CoA reductase) as indicated by direct enzymatic measurements and the absence of inhibition if [¹⁴C]mevalonate was used as a precursor. At high concentrations (above 0.5 mg/mL), inhibition of cholesterol biosynthesis was not only seen at an early step where it increased considerably with dose, but also at later steps resulting in the accumulation of the precursors lanosterol and 7-dehydrocholesterol. No desmosterol was formed which, however, was a major precursor accumulating in the presence of triparanol. Thus, the accumulation of sterol precursors seem to be of less therapeutic significance during consumption of garlic, because it requires concentrations one or two orders of magnitude above those affecting HMG-CoA reductase. Alliin, the main sulfur-containing compound of garlic, was without effect itself. If converted to allicin, it resulted in similar changes of the sterol pattern. This suggested that the latter compound might contribute to the inhibition at the late steps. In contrast, nicotinic acid and particularly adenosine caused moderate inhibition of HMG-CoA reductase activity and of cholesterol biosynthesis suggesting that these compounds participate, at least in part, in the early inhibition of sterol synthesis by garlic extracts. Dedication: This article is dedicated to Prof. Dr. D. Mecke on the occasion of his 60th birthday.     

Platinum and Palladium Organometallic Compounds: Disrupting the Ergosterol Pathway in Trypanosoma cruzi

Current treatment for Chagas’ disease is based on two drugs, Nifurtimox and Benznidazol, which have limitations that reduce the effectiveness and continuity of treatment. Thus, there is an urgent need to develop new, safe and effective drugs. In previous work, two new metal-based compounds with trypanocidal activity, Pd-dppf-mpo and Pt-dppf-mpo, were fully characterized. To unravel the mechanism of action of these two analogous metal-based drugs, high-throughput omics studies were performed. A multimodal mechanism of action was postulated with several candidates as molecular targets. In this work, we validated the ergosterol biosynthesis pathway as a target for these compounds through the determination of sterol levels by HPLC in treated parasites. To understand the molecular level at which these compounds participate, two enzymes that met eligibility criteria at different levels were selected for further studies: phosphomevalonate kinase (PMK) and lanosterol 14-α demethylase (CYP51). Molecular docking processes were carried out to search for potential sites of interaction for both enzymes. To validate these candidates, a gain-of-function strategy was used through the generation of overexpressing PMK and CYP51 parasites. Results here presented confirm that the mechanism of action of Pd-dppf-mpo and Pt-dppf-mpo compounds involves the inhibition of both enzymes.     

Expanding the Binding Envelope of CYP51 Inhibitors Targeting Trypanosoma cruzi with 4‐Aminopyridyl‐Based Sulfonamide Derivatives

Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4‐aminopyridyl‐based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub‐nanomolar compound 3 , have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å X‐ray structure, TcCYP51 is in a conformation that has been influenced by compound 3 and is distinct from the previously characterized ground‐state conformation of CYP51 drug–target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template.     

Production and Application of Novel Sterol Esterases from <Emphasis Type="Italic">Aspergillus</Emphasis> Strains by Solid State Fermentation

Among numerous mesophilic fungi screened for sterol esterase activity followed by the esterification reaction between plant β-sitosterol and lauric acid in organic solvent, six Aspergillus strains were selected as the most active producers. These fungi had not been studied previously for sterol esterase production. The fungi were cultivated under solid state fermentation (SSF) conditions. The gently dried SSF cultures as such were tested in the esterification reactions, without any special enzyme isolation and purification (downstream) processes. All the six Aspergillus SSF preparations were able to synthesize sterol esters. Sterol esterase activity of these GRAS cleared Aspergillus strains was inducible by sterol ester supplementation to the SSF medium and showed remarkably different moisture optimum during growth as compared to the production of lipase (determined by pNP-palmitate). Genome analysis revealed that sterol esterase production might be a common feature of many Aspergillus species. The synthetic usefulness of the best SSF preparations of A. oryzae NRRL 6270 and A. sojae NRRL 6271 was demonstrated by synthesis of esters of plant sterols with lauric acid resulting in 45–63% conversions (GC) and 27–38% isolated yields of steryl laurates. The isomer preference of A. oryzae NRRL 6270 towards the 10E,12Z isomer of conjugated linoleic acid (CLA) in the esterification reaction with plant sterols was also determined.     

Side-chain oxysterol regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A reductase activity

Side-chain oxysterols are known to be potent inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase, a key regulatory enzyme in the biosynthesis of sterols. Structural variations in the side-chain oxysterols influence enzyme inhibition. Under certain conditions, biological systems have been induced to produce side-chain oxysterols, adding support to the hypothesis that oxysterols may be natural regulators of sterol biosynthesis in the intact cell. Specific inhibition of sterol biosynthesis is of interest as it may prove useful in the prevention or reversal of various cardiovascular disease states, as well as in the control of normal and abnormal cell growth. Based on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids”, Atlanta, Georgia, May 1994.     

Squalene – biochemistry,molecular biology,process biotechnology,and applications

Squalene is a natural triterpene and an important intermediate of sterol and hopanoid biosynthesis in various types of cell from bacteria to human. Synthesis and further conversion of squalene are key steps in the metabolism of sterols and related components. Here we summarize the recent knowledge of squalene biochemistry, its molecular properties, and its physiological effects. We compare squalene biosynthetic pathways in different cell types and describe biotechnological strategies to isolate this lipid. Finally, applications of squalene in nutrition, pharmacy, and medicine are discussed.     

Sterols in yeast subcellular fractions

Yeast is the most primitive organism synthesizing substantial amounts of sterols. Because of this eucaryotic organism's versatility in growth conditions, ease of culture, well-defined genetic mechanism, and characteristic subcellar architecture, it is readily applied to studies of the role of sterols in the general economy of the cell. Sterols exist in two major form, as the free sterol, or esterified with long chain fatty acids. The importance of sterols for this organism can be demonstrated using a naturally occurring antimycotic azasterol. This agent inhibits yeast growth. Three effects are seen on sterol synthesis: inhibition of the enzymes Δ¹⁴-reductase, sterol methyltransferase, and methylene reductase. Cells cultured on respiratory substrates are more sensitive to inhibition than are cells growing on glucose. We have demonstrated a relationship between respiratory competency and sterol biosynthesis in this organism. Many mutants altered in sterol synthesis are respirationally defective and must growth fermentatively. One clone has temperature conditional respiration. Experiments with purified mitochondria, perpared from this mutant and its isogenic wildtype, show that the mutant organism is able to respire at the higher temperature but lacks the ability to couple respiration to phosphorylation. No similar loss is seen in the wild-type clones. Data are given which support the proposal that, for inclusion in mitochondrial structures, yeast cells may discriminate among sterols available from the total sterol pool in favor of ergosterol.     

In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.     

The inhibitory effect of neem (Azadirachta indica) leaf extracts on aflatoxin synthesis inAspergillus parasiticus

The effect of neem (Azadirachta indica) leaf extracts onAspergillus parasiticus growth and aflatoxin biosynthesis was investigated. The extracts were prepared by blending 50 g (wet weight) of fresh leaves in one 1 of 10 mM potassium phosphate (pH 7.0) or by boiling the leaves in the buffer. Extracts were added to fungal growth media at 1, 5, 10, 20 and 50% (vol/vol) concentrations prior to inoculation. The formulations did not affect fungal growth (i.e., mycelial dry weight) but essentially blocked (>98%) aflatoxin biosynthesis at concentrations greater than 10% (vol/vol). The inhibitory effect was somewhat diminished (60–70%) inhibition) in heated leaf extracts. Volatile components of the extracts were analyzed using capillary gas chromatography/mass spectrometry; the major volatile component was 3-methyl 2-buten-1-ol. However, volatiles from blended leaf extracts did not affect either aflatoxin synthesis or fungal growth. The neem-mediated inhibition appears to involve regulation of secondary metabolism, because once secondary biosynthesis was initiated the inhibitory effect of the neem leaf constituents was lost. Presented at the AOCS meeting in New Orleans in May 1987.     

Growth ofCururbita maxima L. plants in the presence of the cycloartenol synthase inhibitor U18666A1

Squash, like other Cucurbitaceae, have unique sterol profiles that offer an excellent opportunity to examine the relationship between sterol biosynthesis and plant growth. To determine the effect of sterol biosynthesis inhibition on squash growth, Cucurbita maxima seedlings with and without cotyledons were subjected to increasing concentrations of the cycloartenol synthase (EC 5.4.99.8) inhibitor 3β-(2-diethylaminoethoxy) androstenone (U18666A). Inhibition of shoot growth was concentration-dependent (from 0, 2, 5, 10, and 20 μM); plants with intact cotyledons grew to 26.4, 23.7, 21.6, 20.0, and 15.6 cm, respectively, at the above inhibitor concentrations, compared to 25.5, 19.4, 17.0, 12.0, and 11 cm for plants with severed cotyledons. In plants with severed cotyledons, 10 and 20 μM U18666A caused rapid necrosis of the first two, newly emerged, primary leaves, and halted new leaf formation. Secondary root formation was initially affected at all inhibitor concentrations regardless of whether cotyledons were present or not. Vegetative tissue showed a decrease in the accumulation of the major squash sterol, 7, 22-stigmastadienol, accompanied by increased accumulation of minor sterol components. Sterol profiles in cotyledons were unaltered. The data show that sterols are crucial for maintaining plant growth and viability, but do not address the cotyledonary effect on growth with respect to sterol biosynthesis. Based on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids”, Atlanta, Georgia, May 1994.     

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-2-ethylhexyl phthalate

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the rat. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-¹⁴C and mevalonate-5-³H into squalene, C₃₀ sterols, and C₂₇ sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of³H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% of control values in 11 days, whereas¹⁴C-acetate conversion was reduced to ca. 35% of control values during the same period.³H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as¹⁴C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from¹⁴C-acetate and³H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were simialr in control and DEHP-fed rats.     

Effects of triparanol and AY-9944 upon sterol biosynthesis inChlorella

The growth rates of three species ofChlorella were inhibited by triparanol and AY-9944.Chlorella emersonii was noticeably more resistant to both inhibitors than the other species. A large number of sterols were isolated and identified in inhibited cultures. Ca. 18 of these were identified from nature for the first time. Triparanol resulted in inhibition of the removal of the 14α methyl group. It also inhibited the second alkylation of the side chain and, in one species, strongly inhibited the Δ⁸ → Δ⁷ isomerase reaction. InC. ellipsoidea, triparanol also inhibited Δ¹⁴-reductase and Δ⁷-reductase. The introduction of the Δ²² double bond was inhibited by both drugs. The effect of AY-9944 was similar to that of triparanol inC. emersonii, but it was an extremely effective Δ¹⁴-reductase inhibitor inC. ellipsoidea. These various types of inhibition of sterol synthesis indicate a lack of specificity of both drugs inChlorella and suggest that primitive plants such as these may be valuable as test organisms in an evaluation of the activity of potential inhibitors of sterol biosynthesis.     

Accumulation of ergosta-8,14-dien-3β-ol bySaccharomyces cerevisiae cultured with an azasterol antimycotic agent

15-Aza-24-methylene-D-homocholesta-8,14-dien-3β-ol, an antimycotic agent, at a concentration of 75 ng/ml inhibited ergosterol biosynthesis inSaccharomyces cerevisiae strain 3701 B resulting in the accumulation of an unusual sterol. Experimental data presented indicate that this sterol is ergosta-8,14-dien-3β-ol. The accumulation of the compound is supportive of current models of biosynthetic pathways for sterols in yeast and is consistent with inhibition by the azasterol of the Δ¹⁴ sterol reductase.     

Syntheses of Ring C Oxysterols: Inhibitors of Sterol Biosynthesis

Oxygenated derivates of cholesterol and lanosterol, known as oxysterols, have consistently displayed significant activity as inhibitors of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase, a key regulatory enzyme in sterol biosynthesis. We have developed the chemical syntheses of ring C oxysterols for evaluation as inhibitors of sterol biosynthesis. A key intermediate in the chemical synthesis was 3beta-benzoyloxy-9alpha, 1alpha-epoxy-5alpha-cholest-7-ene (1), whose structure was confirmed by X-ray crystallographic analysis and is presented herein.