Inhibitors Of Sterol Synthesis Research Articles

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.

Transcriptome analysis reveals that high temperatures alter modes of lipid metabolism in juvenile turbot (Scophthalmus maximus) liver

With the increase in farming density and the continuously high summer temperatures against the background of global warming, high temperature stress has become a major challenge in fish farming. In this study, we simulated the high temperature environments (20 °C, 24 °C, and 28 °C) that may occur during turbot culture. High-throughput sequencing was used to investigate the lipid metabolism response patterns in juvenile turbot liver under high temperature stress. A total of 2067 differentially expressed genes (DEGs) were identified. KEGG analysis revealed that the DEGs were mainly associated with glycerophospholipid metabolism, steroid biosynthesis, glycerolipid metabolism, fatty acid metabolic pathways, and the PPAR signaling pathway. A regulatory network was constructed to further elucidate the transcriptional regulation of lipid metabolism. We speculated that high temperature activates PPAR signaling pathway through interaction with ligands such as fatty acids. On the one hand, the HMGCS1 gene in this pathway can inhibit sterol synthesis by down-regulating the expression of key genes in steroid biosynthesis pathway (SQLE, EBP, and DHCR24). On the other hand, the expression of ACSL1 in this pathway is significantly increased under high temperature, which may play an important role in regulating fatty acid metabolism. Moreover, we collected blood and detected changes in serum lipid parameters; the variation patterns were also consistent with our results. These findings reveal that lipid metabolism has an important regulatory role in stress resistance when turbot is exposed to high temperatures.

The first study of triazole fungicide difenoconazole oxidation and its voltammetric and flow amperometric detection on boron doped diamond electrode

Triazole pesticides act as sterol synthesis inhibitors and have been considered as redox-inactive with a variety of electrode materials. In this study, electrochemical oxidation of the fungicide difenoconazole (DFC) was studied in aqueous solutions on oxygen terminated boron doped diamond electrode. It provided a pH-independent irreversible anodic peak at a very positive potential of ca +1.75 V (vs. Ag/AgCl) in acidic media, which could be used for its quantitation in batch voltammetry and flow injection analysis with amperometric detection. The attained detection limits for the determination of DFC in 0.1 mol L−1 HNO3 / phosphate buffer pH 2.0 were 0.049 μmol L−1 for differential pulse voltammetry and 1.58 μmol L−1 for FIA-ED. The former method did not suffer from any significant matrix interferences or interferences of other fungicides and yielded acceptable percentage recovery values (ranging from 96% to 107%) for DFC determination in model river water samples. Similar results comparing both methods at a 95% confidence level were obtained for DFC determination in pesticide preparations. Further, an oxidation mechanism leading to hydroxylated derivative of DFC and subsequent formation of a quinone was suggested. Results were supported with HOMO spatial distribution calculations and atomic charges calculations for arising radical cations.

Lathosterol Oxidase (Sterol C-5 Desaturase) Deletion Confers Resistance to Amphotericin B and Sensitivity to Acidic Stress in Leishmania major.

Lathosterol oxidase (LSO) catalyzes the formation of the C-5-C-6 double bond in the synthesis of various types of sterols in mammals, fungi, plants, and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C-5-C-6 desaturation, we generated an LSO-null mutant line (lso- ) in Leishmania major, the causative agent for cutaneous leishmaniasis. lso- parasites lacked the ergostane-based sterols commonly found in wild-type L. major and instead accumulated equivalent sterol species without the C-5-C-6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso- mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alterations in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania All these defects in lso- were corrected upon the restoration of LSO expression. Together, these findings suggest that the C-5-C-6 double bond is vital for the structure of the sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress.IMPORTANCE Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5-C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5-C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.

1357. A Combination of Itraconazole and Amiodarone Is Highly Effective Against Trypanosoma cruzi Infection of Human Stem Cell-Derived Cardiomyocytes

BackgroundTrypanosoma cruzi is the etiologic agent of Chagas disease, which can result in severe cardiomyopathy. Trypanosoma cruzi is endemic to the Americas, and of particular importance in Latin America. In the United States and other nonendemic countries, rising case numbers have been observed. The only drugs available so far are benznidazole and nifurtimox, which have limited efficacy during chronic infection. We repurposed itraconazole, originally an antifungal, in combination with amiodarone, an antiarrhythmic, with the goal to interfere with Tc infection. Both drugs inhibit sterol synthesis, while amiodarone also inhibits calcium metabolism of Trypanosoma cruzi.MethodsHuman pluripotent stem cells (HiPSC) were differentiated to cardiomyocytes (HiPSC-CM). Vero cells or HiPSC-CM were infected with the T. cruzi trypomastigotes Y strain in the presence of itraconazole and/or amiodarone. After 48 hours, infection and multiplication were evaluated by Giemsa stain. Benznidazole was used as a reference compound. Cell viability was verified by XTT assay.ResultsItraconazole and amiodarone showed dose-dependent interference with T. cruzi infection of Vero cells or HiPSC-CM. The combination of itraconazole and amiodarone was more potent than the single substances, or benznidazole at therapeutic concentrations, without affecting host cell metabolism. In addition to effects on infection, itraconazole, or amiodarone affected T. cruzi multiplication. Here, itraconazole/amiodarone combinations were more potent than either alone, both, in Vero cells, and HiPSC-CM.ConclusionOur in vitro data suggest that a combination of itraconazole and amiodarone might serve as an effective new treatment option for Chagas disease, particularly cardiac involvement, in human and animal patients.Disclosures All authors: No reported disclosures.

Sterol methyltransferase is required for optimal mitochondrial function and virulence in Leishmania major.

Limited knowledge on the exact functions of ergostane-based sterols has hampered the application of sterol synthesis inhibitors against trypanosomatid parasites. Sterol methyltransferase (SMT) is directly involved in the synthesis of parasite-specific C24-methylated sterols, including ergosterol and 5-dehydroepisterol. While pharmacological studies hint at its potential as a drug target against trypanosomatids, direct evidence for the cellular function and essentiality of SMT is lacking. Here, we characterized the SMT knockout mutants and their complemented strains in Leishmania major, the causative agent for cutaneous leishmaniasis. Deletion of SMT alleles led to a complete loss of C24-methylated sterols, which were replaced by cholestane-based sterols. SMT-null mutants were fully viable and replicative in culture but showed increased sensitivity to sphingolipid synthesis inhibition. They were not particularly vulnerable to heat, acidic pH, nitrosative or oxidative stress, yet exhibited high mitochondrial membrane potential and increased superoxide generation indicating altered physiology of the mitochondria. Despite possessing high levels of GPI-anchored glycoconjugates, SMT-null mutants showed significantly attenuated virulence in mice. In total, our study reveals that the biosynthesis of ergostane-based sterols is crucial for the proper function of mitochondria and the proliferation of Leishmania parasites in mammals.

Antifungal Activity of the Lipophilic Antioxidant Ferrostatin-1.

Ferrostatin-1 (Fer-1) is a lipophilic antioxidant that effectively blocks ferroptosis, a distinct non-apoptotic form of cell death caused by lipid peroxidation. During many infections, both pathogens and host cells are subjected to oxidative stress, but the occurrence of ferroptosis had not been investigated. We examined ferroptosis in macrophages infected with the pathogenic yeast Histoplasma capsulatum. Unexpectedly, Fer-1 not only reduced the death of macrophages infected in vitro, but inhibited the growth of H. capsulatum and related species Paracoccidioides lutzii and Blastomyces dermatitidis at concentrations under 10 μm. Other antioxidant ferroptosis inhibitors, including liproxstatin-1, did not prevent fungal growth or reduce macrophage death. Structural analysis revealed a potential similarity of Fer-1 to inhibitors of fungal sterol synthesis, and ergosterol content of H. capsulatum decreased more than twofold after incubation with Fer-1. Strikingly, additional Fer-1 analogues with slight differences from Fer-1 had limited impact on fungal growth. In conclusion, the ferroptosis inhibitor Fer-1 has unexpected antifungal potency distinct from its antiferroptotic activity.

Synthetic antimicrobial peptides of the halictines family disturb the membrane integrity of Candida cells

We compared the potency of four derivatives of the antimicrobial peptide halictine-2 against six Candida species. Observed activity was peptide and species specific. Halictines rapidly permeabilized cell membranes and caused the leakage of cytosolic components. Their killing potential was enhanced by the commercial antimicrobial agent octenidine dihydrochloride. The effect on C. glabrata cells did not depend on the activity of Cdr pumps, but was influenced by their lipid composition. The pre-treatment of cells with myriocin, an inhibitor of sphingolipid synthesis, enhanced the peptides' activity, whereas pre-treatment with terbinafine and fluconazole, inhibitors of sterol synthesis, significantly weakened their efficacy. The killing efficacy of peptides increased in combination with amphotericin B. Thus the mode of action of halictines is likely to depend on the plasma-membrane sterols, which might explain the observed differences among the tested Candida species.

Sterol biosynthesis is required for heat resistance but not extracellular survival in leishmania.

Sterol biosynthesis is a crucial pathway in eukaryotes leading to the production of cholesterol in animals and various C24-alkyl sterols (ergostane-based sterols) in fungi, plants, and trypanosomatid protozoa. Sterols are important membrane components and precursors for the synthesis of powerful bioactive molecules, including steroid hormones in mammals. Their functions in pathogenic protozoa are not well characterized, which limits the development of sterol synthesis inhibitors as drugs. Here we investigated the role of sterol C14α-demethylase (C14DM) in Leishmania parasites. C14DM is a cytochrome P450 enzyme and the primary target of azole drugs. In Leishmania, genetic or chemical inactivation of C14DM led to a complete loss of ergostane-based sterols and accumulation of 14-methylated sterols. Despite the drastic change in lipid composition, C14DM-null mutants (c14dm −) were surprisingly viable and replicative in culture. They did exhibit remarkable defects including increased membrane fluidity, failure to maintain detergent resistant membrane fraction, and hypersensitivity to heat stress. These c14dm − mutants showed severely reduced virulence in mice but were highly resistant to itraconazole and amphotericin B, two drugs targeting sterol synthesis. Our findings suggest that the accumulation of toxic sterol intermediates in c14dm − causes strong membrane perturbation and significant vulnerability to stress. The new knowledge may help improve the efficacy of current drugs against pathogenic protozoa by exploiting the fitness loss associated with drug resistance.

Starvation of low-density lipoprotein-derived cholesterol induces bradyzoite conversion in Toxoplasma gondii.

BackgroundLacking enzymes for sterol synthesis, the intracellular protozoan Toxoplasma gondii scavenges cholesterol from host cells to multiply. T. gondii has a complex life cycle consisting of two asexual stages; the proliferative stage (tachyzoite), and the latent stage characterized by tissue cysts (bradyzoite). In vitro, bradyzoite development can be induced by mimicking host immune response stressors through treatment with IFN-γ, heat shock, nitric oxide, and high pH. However, the extent to which host nutrients contribute to stage conversion in T. gondii is unknown. In this study, we examined the impact of host cholesterol levels on stage conversion in this parasite.MethodsGrowth of T. gondii tachyzoites (ME49 strain) was investigated in Chinese hamster ovary (CHO) cells using various concentrations of low-density lipoprotein (LDL), oleic acid, or glucose. Squalestatin, which is an inhibitor of squalene synthase and is, therefore, an inhibitor of sterol synthesis, was used to treat the CHO cells. Tachyzoite to bradyzoite conversion rates were analyzed by indirect fluorescent antibody tests.ResultsParasite growth was significantly enhanced by addition of exogenous LDL, whereas no such enhancement occurred with oleic acids or glucose. In ME49, growth inhibition from squalestatin treatment was not obvious. Although growth of the RH strain was unaffected by squalestatin in the presence of lipoprotein, in its absence growth of this strain was suppressed. The frequency of BAG1-positive vacuoles in ME49 increased under lipoprotein-free conditions. However, addition of exogenous LDL did not increase tachyzoite to bradyzoite conversion in this strain. Furthermore, treatment with squalestatin did not enhance stage conversion.ConclusionOur results suggest that LDL-derived cholesterol levels play a crucial role in bradyzoite conversion in T. gondii.

Effect of Terbinafin and DMSO on The Gene Expression Level of Squalene Synthase (Sqs) and Amorpha-4,11-Diene Synthase (Ads) in Artemisia Annua L

Artemisinin, a secondary metabolite from Artemisia annua L. is a sesquiterpene lactone that has antimalaria activity but produced at low quantities by the plant. Low levels of artemisinin in the plant is related to the biosynthetic pathways influenced by specific enzymes that play role in the formation of artemisinin. Farnesyl diphosphate (FDP), which is the main precursor of artemisinin, also known as the precursor for the formation of sterols. Compared with the other compounds, sterol biosynthetic pathway is the biggest competitor of the artemisinin production since sterols are needed by plants to regulate membrane fluidity and permeability. This research aimed to study the effect of terbinafin and DMSO as sterol synthesis inhibitors on the regulation of the artemisinin biosynthetic pathway by analyzing the expression level of two genes linked, squalene synthase (SQS) and amorpha-4,11-diene synthase (ADS) using quantitative PCR (qPCR) and the amount of artemisinin is determined using high performance liquid chromatography (HPLC). The results showed that at mRNA levels, terbinafin 30μM had no significant effect on SQS and ADS expression levels, but it increased the amount of artemisinin at 50hours incubation up to 1.36 times higher than control. DMSO increased the expression level of ADS up to 3-4 times and increased the content of artemisinin up to 2.42 times higher than control at 50hours incubation time

Feed restriction, but not l-carnitine infusion, alters the liver transcriptome by inhibiting sterol synthesis and mitochondrial oxidative phosphorylation and increasing gluconeogenesis in mid-lactation dairy cows

Abomasal carnitine infusion during acute feed restriction increases hepatic fatty acid oxidation and decreases liver lipid in dairy cows. Eight mid-lactation Holstein cows were used in a replicated 4×4 Latin square design with 14-d periods. A 2×2 factorial arrangement was used to determine the effects of water infusion+ad libitum dry matter intake (DMI), water infusion+restricted DMI (50% of previous 5-d average), l-carnitine infusion (20g/d)+ad libitum DMI, or l-carnitine infusion+restricted DMI. Liver RNA from 7 healthy cows was used for transcriptome profiling using a bovine microarray. An ANOVA with a false discovery rate was used to identify treatment and interaction effects. A substantial transcriptome change was observed only with DMI restriction, resulting in 312 (155 downregulated, 157 upregulated) differentially expressed genes. Quantitative PCR was performed to verify microarray data and measure expression of additional genes not present on the microarray. The quantitative PCR data confirmed the effect of feed restriction but not of l-carnitine treatment. Feed restriction increased expression of GPX3 and of genes associated with gluconeogenesis (PC, PDK4), inflammation (SAA3), and signaling (ADIPOR2). In contrast, feed restriction downregulated BBOX, a key for l-carnitine biosynthesis, and the transcription factor HNF4A. The bioinformatics functional analysis of genes affected by DMI restriction uncovered biosynthesis of cholesterol and energy generation by mitochondrial respiration as the most relevant and inhibited functions. The data also indicated an increase of flux toward gluconeogenesis. We interpreted those results as a likely response of the liver to spare energy and provide glucose for the lactating mammary gland during feed deprivation.

Chronic phase of Chagas disease: why should it be treated? A comprehensive review

The pathogenesis and evolutive pattern of Chagas disease suggests that the chronic phase should be more widely treated in order to (i) eliminate Trypanosoma cruzi and prevent new inflammatory foci and the extension of tissue lesions, (ii) promote tissue regeneration to prevent fibrosis, (iii) reverse existing fibrosis, (iv) prevent cardiomyopathy, megaoesophagus and megacolon and (v) reduce or eliminate cardiac block and arrhythmia. All cases of the indeterminate chronic form of Chagas disease without contraindications due to other concomitant diseases or pregnancy should be treated and not only cases involving children or recently infected cases. Patients with chronic Chagas cardiomyopathy grade II of the New York Heart Association classification should be treated with specific chemotherapy and grade III can be treated according to medical-patient decisions. We are proposing the following new strategies for chemotherapeutic treatment of the chronic phase of Chagas disease: (i) repeated short-term treatments for 30 consecutive days and interval of 30-60 days for six months to one year and (ii) combinations of drugs with different mechanisms of action, such as benznidazole + nifurtimox, benznidazole or nifurtimox + allopurinol or triazole antifungal agents, inhibition of sterol synthesis.

ChemInform Abstract: Inhibitors of Sterol Synthesis. An Efficient and Specific Side Chain Oxidation of 3β-Hydroxy-5α-cholest-8(14)-en-15-one. Facile Access to Its Metabolites and Analogs.

ChemInform Abstract: Inhibitors of Sterol Synthesis. An Efficient and Specific Side Chain Oxidation of 3β-Hydroxy-5α-cholest-8(14)-en-15-one. Facile Access to Its Metabolites and Analogs.

Effect of Miconazole and Terbinafine on Artemisinin Content of Shooty Teratoma of Artemisia annua

The effects produced by the addition of sterol synthesis inhibitors on the artemisinin content of the transgenic organ culture (A. tumefaciens ATCC 33970 or 15955) of Artemisia annua are presented. The transgenic tissue produced 3-4 fold higher levels of artemisinin 0.84% (56.3 mg/L) within a short culture period compared with field grown plants (0.23%). The addition of the sterol synthesis inhibitors, miconazole and terbinafine, to these transgenic cultures resulted in enhanced artemisinin content up to 1.15% and 1.44%, respectively. Further enhancement of artemisinin content was achieved by varying the addition time of the sterol synthesis inhibitor to the cultures. The best artemisinin content (2.62%) was observed after terbinafine (10 mg/L) addition on the sixteenth day of the culture period.

Cryptococcus neoformans Site‐2 protease is required for virulence and survival in the presence of azole drugs

In the human fungal pathogen Cryptococcus neoformans, the SREBP orthologue Sre1 is important for adaptation and growth in nutrient-limiting host tissues. In this study, we characterize the C. neoformans serotype A Sre1 and its activating protease, Stp1. We demonstrate that Stp1 is a functionally conserved orthologue of the mammalian Site-2 protease and that Stp1 cleaves Sre1 within its predicted first transmembrane segment. Gene expression analysis revealed that Stp1 is required for both Sre1-dependent and Sre1-independent gene transcription, indicating that other substrates of Stp1 may exist. Using gas chromatography, we showed that Sre1 and Stp1 are required for both normoxic and hypoxic ergosterol biosynthesis, and therefore cells lacking SRE1 or STP1 are defective for growth in the presence of low levels of the ergosterol biosynthesis inhibitors, itraconazole and 25-thialanosterol. Importantly, our studies demonstrated fungicidal effects of itraconazole and 25-thialanosterol towards sre1Delta and stp1Delta cells, demonstrating that the Sre1 pathway is required for both growth and survival in the presence of sterol biosynthesis-inhibiting antifungal drugs. Given the need for fungicidal drugs, we propose that inhibitors of Stp1, Sre1, or other regulators of Sre1 function administered in combination with a sterol synthesis inhibitor could prove an effective anticryptococcal therapy.

Role of MGA2 in regulation of lipid metabolism in Saccharomyces cerevisiae

The Saccharomyces cerevisiae MGA2 gene encodes a protein that is an important regulator of lipid metabolism in the cell. Mga2p plays a major role in unsaturated fatty acid production through its regulation of the transcription and mRNA stability of OLE1, whose product is the Δ‐9 fatty acid desaturase. We have found that mga2Δ strains also contain elevated relative amounts of squalene, a sterol pathway intermediate, when compared to wild‐type cells. A possible explanation for the higher squalene levels detected in mga2Δ cells is lower expression of the ERG1 gene. ERG1 encodes squalene epoxidase, which catalyzes the conversion of squalene to squalene epoxide. To explore whether MGA2 plays a role in regulating ERG1 expression, we first examined effects on ERG1 transcription using a promoter‐lacZ reporter gene. We report here that in addition to MGA2's role in regulation of unsaturated fatty acids, MGA2 is required for full basal expression of ERG1. In contrast to OLE1, we found that ERG1 expression was not repressed by unsaturated fatty acids. However, ERG1 expression was elevated in response to miconazole, an inhibitor of sterol synthesis in yeast

Lipids

We have recently shown that an inhibition of sterol synthesis by fenpropimorph leads to an accumulation of sterol precursors, hydroxypalmitic acid-containing glucosylceramides and detergent resistant membranes in the Golgi bodies instead of the plasma membrane, suggesting that the individual molecules or the microdomains were blocked in the Golgi. These results and others from several eukaryotic models link lipid metabolism with membrane morphodynamics that are involved in membrane trafficking. Focus has been expanded to other lipid families, and numerous evidences are given showing lipids and lipid-modifying enzymes as key regulators of membrane homeostasis which can strongly regulate membrane morphodynamics and therefore trafficking. Beside protein-based machineries, lipid-based machineries are also shown as crucial regulatory forces involved in protein transport and sorting.

Inhibition of the Enzymatic Activity of Heme Oxygenases by Azole-Based Antifungal Drugs

Ketoconazole (KTZ) and other azole antifungal agents are known to have a variety of actions beyond the inhibition of sterol synthesis in fungi. These drugs share structural features with a series of novel heme oxygenase (HO) inhibitors designed in our laboratory. Accordingly, we hypothesized that therapeutically used azole-based antifungal drugs are effective HO inhibitors. Using gas chromatography to quantify carbon monoxide formation in vitro and in vivo, we have shown that azole-containing antifungal drugs are potent HO inhibitors. Terconazole, sulconazole, and KTZ were the most potent drugs with IC(50) values of 0.41 +/- 0.01, 1.1 +/- 0.4, and 0.3 +/- 0.1 microM for rat spleen microsomal HO activity, respectively. Kinetic characterization revealed that KTZ was a noncompetitive HO inhibitor. In the presence of KTZ (2.5 and 10 microM), K(m) values for both rat spleen and brain microsomal HO were not altered; however, a significant decrease in the catalytic capacity (V(max)) was observed (P < 0.005). KTZ was also found to weakly inhibit nitric-oxide synthase with an IC(50) of 177 +/- 2 microM but had no effect on the enzymatic activity of NADPH cytochrome P450 reductase. Because these drugs were effective within the concentration range observed in humans, it is possible that inhibition of HO may play a role in some of the pharmacological actions of these antimycotic drugs.

Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells

Sterol synthesis is required for Sonic hedgehog (Shh) signal transduction. Errors in Shh signal transduction play important roles in the formation of human tumors, including medulloblastoma (MB). It is not clear which products of sterol synthesis are necessary for Shh signal transduction or how they act. Here we show that cholesterol or specific oxysterols are the critical products of sterol synthesis required for Shh pathway signal transduction in MB cells. In MB cells, sterol synthesis inhibitors reduce Shh target gene transcription and block Shh pathway-dependent proliferation. These effects of sterol synthesis inhibitors can be reversed by exogenous cholesterol or specific oxysterols. We also show that certain oxysterols can maximally activate Shh target gene transcription through the Smoothened (Smo) protein as effectively as the known Smo full agonist, SAG. Thus, sterols are required and sufficient for Shh pathway activation. These results suggest that oxysterols may be critical regulators of Smo, and thereby Shh signal transduction. Inhibition of Shh signaling by sterol synthesis inhibitors may offer a novel approach to the treatment of MB and other Shh pathway-dependent human tumors.