Inhibition Of Sterol Research Articles

Chemical Inhibition of Sterol Biosynthesis.

Cholesterol is an essential molecule of life, and its synthesis can be inhibited by both genetic and nongenetic mechanisms. Hundreds of chemicals that we are exposed to in our daily lives can alter sterol biosynthesis. These also encompass various classes of FDA-approved medications, including (but not limited to) commonly used antipsychotic, antidepressant, antifungal, and cardiovascular medications. These medications can interfere with various enzymes of the post-lanosterol biosynthetic pathway, giving rise to complex biochemical changes throughout the body. The consequences of these short- and long-term homeostatic disruptions are mostly unknown. We performed a comprehensive review of the literature and built a catalogue of chemical agents capable of inhibiting post-lanosterol biosynthesis. This process identified significant gaps in existing knowledge, which fall into two main areas: mechanisms by which sterol biosynthesis is altered and consequences that arise from the inhibitions of the different steps in the sterol biosynthesis pathway. The outcome of our review also reinforced that sterol inhibition is an often-overlooked mechanism that can result in adverse consequences and that there is a need to develop new safety guidelines for the use of (novel and already approved) medications with sterol biosynthesis inhibiting side effects, especially during pregnancy.

Elucidation of sterol biosynthesis pathway and its co-regulation with fatty acid biosynthesis in the oleaginous marine protist Schizochytrium sp.

Sterols constitute vital structural and regulatory components of eukaryotic cells. In the oleaginous microorganism Schizochytrium sp. S31, the sterol biosynthetic pathway primarily produces cholesterol, stigmasterol, lanosterol, and cycloartenol. However, the sterol biosynthesis pathway and its functional roles in Schizochytrium remain unidentified. Through Schizochytrium genomic data mining and a chemical biology approach, we first in silico elucidated the mevalonate and sterol biosynthesis pathways of Schizochytrium. The results showed that owing to the lack of plastids in Schizochytrium, it is likely to use the mevalonate pathway as the terpenoid backbone pathway to supply isopentenyl diphosphate for the synthesis of sterols, similar to that in fungi and animals. In addition, our analysis revealed a chimeric organization of the Schizochytrium sterol biosynthesis pathway, which possesses features of both algae and animal pathways. Temporal tracking of sterol profiles reveals that sterols play important roles in Schizochytrium growth, carotenoid synthesis, and fatty acid synthesis. Furthermore, the dynamics of fatty acid and transcription levels of genes involved in fatty acid upon chemical inhibitor-induced sterol inhibition reveal possible co-regulation of sterol synthesis and fatty acid synthesis, as the inhibition of sterol synthesis could promote the accumulation of fatty acid in Schizochytrium. Sterol and carotenoid metabolisms are also found possibly co-regulated, as the inhibition of sterols led to decreased carotenoid synthesis through down-regulating the gene HMGR and crtIBY in Schizochytrium. Together, elucidation of the Schizochytrium sterol biosynthesis pathway and its co-regulation with fatty acid synthesis lay the essential foundation for engineering Schizochytrium for the sustainable production of lipids and high-value chemicals.

Design, Synthesis, Antimicrobial and Ergosterol Inhibition Activity of New 4-(Imidazo[1,2-a]Pyridin-2-yl)Quinoline Derivatives

In the search for new lead compounds targeting fungi and bacteria, based on the in silico molecular docking binding affinity analysis against sterol 14-demethylase enzyme, a series of 4-(imidazo[1,2-a]pyridin-2-yl)quinoline (6a-l) have been synthesized by a cyclo condensation reaction of 4-(2-bromoacetyl)quinolin-1-ium bromide (4a-d) with substituted 2-aminopyridine (5a-c). The newly synthesized 4-(imidazo[1,2-a]pyridin-2-yl)quinoline derivatives were characterized by spectroscopic techniques. The imidazo[1,2-a]pyridinyl-quinoline derivatives were studied for ADME analysis and molecular docking against sterol 14-demethylase, enzyme essential in ergostrol biosynthesis in fungi. All compounds were evaluated for in vitro antifungal activity against Candida albicans (NCIM 3100) and Aspergillus niger (ATCC 504) and in vitro antibacterial activity against Escherichia coli (NCIM 2574), Proteus mirabilis (NCIM 2388), Bacillus subtilis (NCIM 2063), Staphylococcus albus (NCIM 2178). Nine 4-(imidazo[1,2-a]pyridin-2-yl)quinoline derivatives 6b, 6d, 6e, 6f, 6g, 6h, 6i, 6j and 6l, reported good antifungal activity against A. niger with MIC 62.5 µg/mL. To substantiate the mode of action compounds 6b, 6e, 6f, 6g and 6h were investigated for ergosterol inhibition assay against A. niger cells sample at 62.5 µg/mL concentration. The sterol inhibition assay analysis revealed that the ergosterol biosynthesis has decreased in the fungal samples treated with the 4-(imidazo[1,2-a]pyridin-2-yl)quinoline derivatives. Thus the substantial antifungal activity of 4-(imidazo[1,2-a]pyridin-2-yl)quinoline derivatives against A. niger suggested that these compounds could aid and assist in the development of lead compounds to treat fungal infections.

Hesperidin Targets Leishmania donovani Sterol C-24 Reductase to Fight against Leishmaniasis.

Hesperidin, a naturally occurring flavanoid, is present in citrus family of fruits. It was found effective against an array of pathogens including fungi, bacteria, viruses, and protozoa. Here, we evaluated its antileishmanial activity and possible mechanism of action through different in vitro and in silico experiments. It inhibited the growth and proliferation of the parasites significantly with a IC50 value of 1.019 ± 0.116 mM in vitro. It also reduced the growth of intra-macrophagic amastigotes with a IC50 value of 0.2858 ± 0.01398 mM. It induced the reactive oxygen species (ROS) in parasites in a dose-dependent manner. Through 2,7-dichloro dihydro fluorescein diacetate (H2DCFDA) staining, it was observed that around 96.9% parasites were ROS positive at 2.0 mM concentration of hesperidin. The ROS generated led to the apoptosis of parasites in a dose-dependent manner as observed by annexin/PI staining. Molecular docking with one of the very important and unique drug-targets of Leishmania donovani sterol C-24 reductase resulted in its significant inhibition. Here, we for the first time showed that hesperidin induced the antileishmanial response through the induction of apoptosis and inhibition of sterol C-24 reductase. Our study will be helpful in the development of a cost-effective antileishmanial lead with higher efficacy.

Synthesis and Biological Evaluation of New 1,2,3‐Triazolyl‐Pyrazolyl‐Quinoline Derivatives as Potential Antimicrobial Agents

AbstractA new series of 4‐{1‐phenyl‐4‐[(4‐phenyl‐1,2,3‐triazol‐1‐yl)methyl]pyrazol‐3‐yl}quinoline (7a‐l) have been synthesized by a click reaction of 4‐(4‐(azidomethyl)‐1‐phenyl‐1H‐pyrazol‐3‐yl)quinoline (5a‐d) with a substituted ethynylbenzene. The newly synthesized 1,2,3‐triazolyl‐pyrazolyl‐quinoline derivatives were evaluated for biological activity against Escherichia coli (NCIM 2574), Proteus mirabilis (NCIM 2388), (Gram negative strains), Bacillus subtilis (NCIM 2063), Staphylococcus albus (NCIM 2178) (Gram positive strains) and in vitro antifungal activity against Candida albicans (NCIM 3100) and Aspergillus niger (ATCC 504). Five 1,2,3‐triazolyl‐pyrazolyl‐quinoline derivatives, 7d, 7 g, 7 h, 7k and 7 l exhibited good antifungal activity against A. niger with MIC 62.5 μg/mL. The compounds 7 h, 7k and 7 l were further evaluated for ergosterol inhibition assay against A. niger cell sample at 62.5 μg/mL concentration. Upon analysis of the sterol inhibition assay, it was revealed that, the ergosterol biosynthesis has decreased in the fungal samples treated with the 1,2,3‐triazolyl‐pyrazolyl‐quinoline derivatives. Thus antimicrobial activity suggested that, these compounds could aid and assist in the development of lead compounds to treat microbial infections.

Synthesis of new thiazolyl-pyrazolyl-1,2,3-triazole derivatives as potential antimicrobial agents

A series of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole derivatives (7a-y) have been synthesized by click reaction of 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-aryl-1,3-thiazole (5a-e) with substituted benzyl azide. The starting compounds 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-aryl-1,3-thiazole (5a-e) were synthesized from corresponding 3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3a-e) by using Ohira-Bestmann reagent. All newly synthesized thiazolyl-pyrazolyl-1,2,3-triazole derivatives were screened for antibacterial activity against two Gram negative strains, Escherichia coli (NCIM 2574), Proteus mirabilis (NCIM 2388), a Gram positive strain Staphylococcus albus (NCIM 2178) and in vitro antifungal activity against Candida albicans (NCIM 3100), Aspergillus niger (ATCC 504) and Rhodotorula glutinis (NCIM 3168). Ten thiazolyl-pyrazolyl-1,2,3-triazole derivatives, 7b, 7g, 7i, 7j, 7k, 7l, 7m, 7n, 7p and 7v exhibited promising antifungal activity against A. niger with MIC 31.5 μg/mL. Compounds 7g, 7i, 7k, 7l and 7m were further evaluated for ergosterol inhibition assay against A. niger cells sample at 31.5 μg/mL concentration. The analysis of sterol inhibition assay revealed that ergosterol biosynthesis is decreased in the fungal samples treated with azole derivatives. Promising antifungal activity suggested that, these compounds could be further promoted for optimization and development which could have the potential to treat against fungal infection.

Dichlorophenyl piperazines, including a recently-approved atypical antipsychotic, are potent inhibitors of DHCR7, the last enzyme in cholesterol biosynthesis

While antipsychotic medications provide important relief from debilitating psychotic symptoms, they also have significant adverse side effects, which might have relevant impact on human health. Several research studies, including ours, have shown that commonly used antipsychotics such as haloperidol and aripiprazole affect cholesterol biosynthesis at the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. This transformation is promoted by the enzyme DHCR7 and its inhibition causes increases in plasma and tissue levels of 7-DHC. The inhibition of this enzymatic step by mutations in the Dhcr7 gene leads to Smith-Lemli-Opitz syndrome, a devastating human condition that can be replicated in rats by small molecule inhibitors of DHCR7. The fact that two compounds, brexpiprazole and cariprazine, that were recently approved by the FDA have substructural elements in common with the DHCR7 inhibitor aripiprazole, prompted us to evaluate the effect of brexpiprazole and cariprazine on cholesterol biosynthesis. We report that cariprazine affects levels of 7-DHC and cholesterol in cell culture incubations at concentrations as low as 5 nM. Furthermore, a common metabolite of cariprazine and aripiprazole, 2,3-(dichlorophenyl) piperazine, inhibits DHCR7 activity at concentrations comparable to those of the potent teratogen AY9944. The cell culture experiments were corroborated in mice in studies showing that treatment with cariprazine elevated 7-DHC in brain and serum. The consequences of sterol inhibition by antipsychotics in the developing nervous system and the safety of their use during pregnancy remains to be established.

AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.

Mechanisms of Anti-Cancer Effects of <i>Vernonia amygdalina</i> Leaf Extract

Problem statement: Vernonia amygdalina (V. amygdalina) has been shown to have cancer cell inhibition and cytotoxic effects. Approach: This study discusses the multi-faceted and multi-linked mechanisms by which cancer tissue inhibition and destruction is achieved by V. amygdalina leaf extract and powder (subsequently called V. amygdalina Extract). Cancer cell inhibition by V. amygdalina is suggested to occur through inhibition of sterol 14-α-demethylase, a microsomal P450-dependent enzyme system) of the membrane of the cancer cell. Inhibition of this enzyme impairs the biosynthesis of ergosterol for the cytoplasmic membrane. Results: This impairment of the synthesis of ergosterol disrupts the close packing of acyl chains of phospholipids and impairs the function of some membrane-bound enzyme systems like ATPase and enzymes of electron-transport system of the cancer cells. V. amygdalina extract inhibits sterol 14-α-demethylase by acting on β3 adrenergic receptors of the cancer cell membranes as a primary ligand in Gi (inhibitory) receptors on cancer β3 lipid cell membrane receptors and mitochondrial ATP energy generation system receptors. Conclusion: By effecting β-oxidation of fatty acids and lipids in cell membranes and cell mitochondrial energy (ATP) generation systems, the V. amygdalina extract uncouples the mitochondrial energy (ATP generation) systems of the cancer cells and cell membranes resulting in the cancer cells being burnt out (consumed) by the extract (when V. amygdalina extract is cytotoxic to the cancer cells) or its functioning being inhibited (when V. amygdalina extract is only inhibitory to the cancer cells.

Requirements for Transitional Endoplasmic Reticulum Site Structure and Function in Saccharomyces cerevisiae

Secretory proteins are exported from the endoplasmic reticulum (ER) at specialized regions known as the transitional ER (tER). Coat protein complex II (COPII) proteins are enriched at tER sites, although the mechanisms underlying tER site assembly and maintenance are not understood. Here, we investigated the dynamic properties of tER sites in Saccharomyces cerevisiae and probed protein and lipid requirements for tER site structure and function. Thermosensitive sec12 and sec16 mutations caused a collapse of tER sites in a manner that depended on nascent secretory cargo. Continual fatty acid synthesis was required for ER export and for normal tER site structure, whereas inhibition of sterol and ceramide synthesis produced minor effects. An in vitro assay to monitor assembly of Sec23p-green fluorescent protein at tER sites was established to directly test requirements. tER sites remained active for approximately 10 min in vitro and depended on Sec12p function. Bulk phospholipids were also required for tER site structure and function in vitro, whereas depletion of phophatidylinositol selectively inhibited coat protein complex II (COPII) budding but not assembly of tER site structures. These results indicate that tER sites persist through relatively stringent treatments in which COPII budding was strongly inhibited. We propose that tER site structures are stable elements that are assembled on an underlying protein and lipid scaffold.

Membrane protein SMP-1 is required for normal flagellum function inLeishmania

Eukaryotic flagella and cilia are surrounded by a membrane that is continuous with, but distinct from, the rest of the plasma membrane. In Leishmania parasites, the inner leaflet of the flagellar membrane is coated with the acylated membrane protein, SMP-1. Here, we provide evidence that SMP-1 stabilizes the flagellar membrane and is required for flagella elongation and function. The expression and flagella targeting of SMP-1 is tightly associated with flagella elongation during amastigote to promastigote differentiation. Deletion of the genes encoding SMP-1 and the flagellar pocket protein SMP-2, led to the production of short flagella and defects in motility. Alterations in the physical properties of the smp-1/smp-2(-/-) flagellar membrane were suggested by: (1) the accumulation of membrane vesicles in the flagellar matrix, and (2) further retraction of flagella following partial inhibition of sterol and sphingolipid biosynthesis. The flagella phenotype of the smp-1/smp-2(-/-) null mutant was reversed by re-expression of SMP-1, but not SMP-2. SMP-1 contains a jelly-roll beta-sheet structure that is probably conserved in all SMP proteins, and forms stable homo-oligomers in vivo. We propose that the SMP-1 coat generates and/or stabilizes sterol- and sphingolipid-rich domains in the flagellar membrane.

Inhibition of sterol 14 α-demethylation of Candida albicans with NND-502, a novel optically active imidazole antimycotic agent

To investigate the mode of action of the newly synthesized optically active imidazole compound, NND-502, (−)-(E)-[4-(2,4-dichlorophenyl)-1,3-dithiolan-2-ylidene]-1-imidazolylacetonitrile, its effect on ergosterol biosynthesis in cell-free extracts of Candidaalbicans was examined and compared with that of the (S)-enantiomer of NND-502 in addition to lanoconazole and bifonazole, both of which are clinically used for the treatment of dermatomycoses. NND-502 was found to interfere with ergosterol biosynthesis by inhibition of sterol 14α-demethylase, while no interference due to the (S)-enantiomer of NND-502 was found, indicating that the stereochemical orientation of the 2,4-dichlorophenyl group plays an important role in the interaction with the enzyme. In terms of drug concentration exerting 50% inhibition of ergosterol biosynthesis, NND-502 was 2·5 and 28 times more effective than that of lanoconazole and bifonazole, respectively.

Stigmasterol reduces plasma cholesterol levels and inhibits hepatic synthesis and intestinal absorption in the rat

Plant sterols compete with cholesterol (cholest-5-en-3 β-ol) for intestinal absorption to limit absorption and lower plasma concentrations of cholesterol. Stigmasterol (24-ethyl-cholesta-5,22-dien-3 β-ol; Δ 22 derivative of sitosterol [24-ethyl-cholest-5-en-3 β-ol]), but not campesterol (24-methyl-cholest-5-en-3 β-ol) and sitosterol, is reported to inhibit cholesterol biosynthesis via inhibition of sterol Δ 24-reductase in human Caco-2 and HL-60 cell lines. We studied the effect of feeding 0.5% stigmasterol on plasma and liver sterols and intestinal cholesterol and sitosterol absorption in 12 wild-type Kyoto (WKY) and 12 Wistar rats. After 3 weeks of feeding, cholesterol and sitosterol absorption was determined in 6 rats from each group by plasma dual-isotope ratio method. After 3 more weeks, plasma and hepatic sterols and hepatic enzyme activities were determined in all rats. After feeding stigmasterol, baseline plasma cholesterol was 1.3 times and plant sterols 3 times greater in WKY compared with Wistar rats. Stigmasterol feeding lowered plasma cholesterol by approximately 11%, whereas plasma campesterol and sitosterol levels were virtually unchanged in both rat strains, and stigmasterol constituted 3.2% of plasma sterols in WKY rats and 1% in Wistar rats. After 6 weeks of feeding, cholesterol and sitosterol absorption decreased 23% and 30%, respectively, in WKY, and 22% and 16%, respectively, in the Wistar rats as compared with untreated rats. The intestinal bacteria in both rat strains metabolized stigmasterol to mainly the 5 β-H stanol (>40%), with only small amounts of 5 α-H derivative (approximately 1.5%), whereas the C-22 double bond was resistant to bacterial metabolism. Hepatic stigmasterol levels increased from 11 μg/g liver tissue to 104 μg/g in WKY rats and from 5 μg/g liver tissue to 21 μg/g in Wistar rats. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity was suppressed 4-fold in the WKY and almost 1.8-fold in Wistar rats, cholesterol 7 α-hydroxylase activity was suppressed 1.6-fold in the WKY and 3.5-fold in Wistar rats, whereas cholesterol 27-hydroxylase activity was unchanged after feeding. In conclusion, stigmasterol, when fed, lowers plasma cholesterol levels, inhibits intestinal cholesterol and plant sterol absorption, and suppresses hepatic cholesterol and classic bile acid synthesis in Wistar as well as WKY rats. However, plasma and hepatic incorporation of stigmasterol is low.

Insig Required for Sterol-mediated Inhibition of Scap/SREBP Binding to COPII Proteins in Vitro*♦

When added to living cells, sterols such as cholesterol and 25-hydroxycholesterol block the lateral movement of sterol regulatory element-binding proteins (SREBPs) into COPII-coated vesicles on endoplasmic reticulum (ER) membranes and thereby prevent the SREBPs from reaching the Golgi complex for processing to the mature forms that activate cholesterol synthesis. Sorting of SREBPs into COPII vesicles is mediated by Sar1 and the coat proteins Sec23 and Sec24. Here, we explore the mechanism of sterol inhibition in vitro through use of protein pull-down assays. We show that addition of cholesterol or 25-hydroxycholesterol to microsomal membranes in vitro blocks Sar1-dependent binding of the Sec23/24 complex to Scap, the SREBP escort protein. This in vitro inhibition is dependent on the presence of Insig-1, an ER resident protein that is necessary for sterol-mediated inhibition of Scap/SREBP transport in intact cells. Sec23/24 binding to Scap requires the hexapeptide sequence MELADL located in a cytoplasmic loop of Scap. This hexapeptide acts as a sterol-regulated ER sorting signal. These studies define the biochemical parameters responsible for regulated sorting of an ER membrane protein into COPII-coated vesicles.

Posaconazole is a potent inhibitor of sterol 14alpha-demethylation in yeasts and molds.

Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata. In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata, Aspergillus fumigatus, and Aspergillus flavus. Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14alpha-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14alpha-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14alpha-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.

Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase.

Azole compounds play a key role as antifungals in agriculture and in human mycoses and as non-steroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad use of azoles is based on their inhibition of certain pathways of steroidogenesis by high-affinity binding to the enzymes sterol 14-alpha-demethylase and aromatase. Sterol 14-alpha-demethylase is crucial for the production of meiosis-activating sterols, which recently were shown to modulate germ cell development in both sexes of mammals. Aromatase is responsible for the physiologic balance of androgens and estrogens. At high doses, azole fungicides and other azole compounds affect reproductive organs, fertility, and development in several species. These effects may be explained by inhibition of sterol 14-alpha-demethylase and/or aromatase. In fact, several azole compounds were shown to inhibit these enzymes in vitro, and there is also strong evidence for inhibiting activity in vivo. Furthermore, the specificity of the enzyme inhibition of several of these compounds is poor, both with respect to fungal versus nonfungal sterol 14-alpha-demethylases and versus other P450 enzymes including aromatase. To our knowledge, this is the first review on sterol 14-alpha-demethylase and aromatase as common targets of azole compounds and the consequence for steroidogenesis. We conclude that many azole compounds developed as inhibitors of fungal sterol 14-alpha-demethylase are inhibitors also of mammalian sterol 14-alpha-demethylase and mammalian aromatase with unknown potencies. For human health risk assessment, data on comparative potencies of azole fungicides to fungal and human enzymes are needed.

In vivo anti-inflammatory effect of statins is mediated by nonsterol mevalonate products.

This study set out to clarify whether the inhibition of sterol or nonsterol derivatives arising from mevalonate biotransformation plays a major role in the in vivo anti-inflammatory action of statins. Hepatic synthesis of all these derivatives was inhibited in mice by administered statins, whereas squalestatin inhibited only sterol derivatives. Using a short-term treatment schedule, we found that statins reduced the hepatic activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase without affecting blood cholesterol. This treatment inhibited lipopolysaccharide- and carrageenan-induced pouch leukocyte recruitment and the exudate production of interleukin-6, monocyte chemotactic protein-1, and RANTES. Coadministration of mevalonate reversed the effect of statin on leukocyte recruitment. The inhibition of sterol synthesis by squalestatin did not have any anti-inflammatory effect, indicating that the biosynthesis of nonsterol compounds arising from mevalonate is crucial for the in vivo regulation of cytokine and chemokine production by statins. Their inhibition by statins may account for the reported anti-inflammatory effects of these drugs and may provide a biochemical basis for the recently reported effects of statins in the prevention of cardiovascular disease and mortality.

Inhibition of sterol 14 alpha-demethylation of Candida albicans with NND-502, a novel optically active imidazole antimycotic agent.

To investigate the mode of action of the newly synthesized optically active imidazole compound, NND-502, (-)-(E)-[4-(2, 4-dichlorophenyl)-1,3-dithiolan-2-ylidene]-1-imidazolylacetonit rile, its effect on ergosterol biosynthesis in cell-free extracts of Candida albicans was examined and compared with that of the (S)-enantiomer of NND-502 in addition to lanoconazole and bifonazole, both of which are clinically used for the treatment of dermatomycoses. NND-502 was found to interfere with ergosterol biosynthesis by inhibition of sterol 14alpha-demethylase, while no interference due to the (S)-enantiomer of NND-502 was found, indicating that the stereochemical orientation of the 2, 4-dichlorophenyl group plays an important role in the interaction with the enzyme. In terms of drug concentration exerting 50% inhibition of ergosterol biosynthesis, NND-502 was 2.5 and 28 times more effective than that of lanoconazole and bifonazole, respectively.

Emopamil-binding Protein, a Mammalian Protein That Binds a Series of Structurally Diverse Neuroprotective Agents, Exhibits Δ8-Δ7 Sterol Isomerase Activity in Yeast

Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae. The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs. A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert delta8-sterol into delta7-sterol, both in vivo and in vitro. In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell. In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains. Our results strongly suggest that EBP and delta8-delta7 sterol isomerase are identical proteins in mammals.