Zaragozic Acid Research Articles

Stereo-controlled Synthesis of Vicinal Tertiary Carbinols: Application in the Synthesis of Zaragozic Acid, Pactamycin and Ryanodol Core Structures.

Here we report a novel and flexible approach for the stereo-controlled synthesis of vicinal tertiary carbinols. The developed strategy featured a highly diastereoselective singlet-oxygen (O21) [4+2] cycloaddition of rationally designed cyclohexadienones (derived from oxidative dearomatization of the corresponding carboxylic-acid appended phenol precursors), followed by programmed "O-O" and "C-C" bond cleavage. In doing so, a highly functionalized and versatile intermediate was identified and prepared in synthetically useful quantity as a plausible precursor to access a variety of designed and naturally occurring vicinal tertiary carbinol containing compounds. Most notably, the developed strategy was successfully applied in the stereo-controlled synthesis of advanced core structures of zaragozic acid, pactamycin and ryanodol.

Pharmacologic inhibition of astrocyte ApoE release

AbstractBackgroundThe ApoE4 allele is the strongest known genetic risk factor for late onset Alzheimer’s disease. Despite this knowledge, therapeutic strategies seeking to modulate brain ApoE homeostasis remain largely unexplored. Several lines of evidence demonstrate that astrocytes function as the primary producers of ApoE within the brain. In this study, we examine how targeted disruption of astrocyte lipid metabolism and protein prenylation effect the secretion of ApoE from primary astrocyte cultures.MethodPrimary cell cultures of mouse astrocytes were grown from 1‐3 day old pups. Drugs were applied to cells in serum free conditions over a range of timepoints and doses. Astrocyte conditioned media was collected from cultures and ApoE and other proteins of interest were measured by western blot.ResultWe find that inhibiting HMG‐CoA reductase, a rate‐limiting enzyme in the cholesterol synthesis pathway, with simvastatin reduces astrocyte ApoE levels in conditioned media in a dose dependent manner, while secretion of other proteins remain preserved. Inhibition of downstream cholesterol synthesis through blockade of the enzyme FDFT1 with zaragozic acid, however, has no effect on total ApoE secretion. Mice with genetic disruption of brain cholesterol metabolism also have similar levels of ApoE in CSF samples compared to control. These data suggest the effect of HMGCR inhibition on ApoE is not mediated through cholesterol. It is known that HMGCR activity is also required for maintenance of cellular protein prenylation. We tested the prenylation inhibitor FGTI‐2734 and found that it is sufficient to block astrocyte ApoE secretion, suggesting that HMGCR inhibitors may exert their effect on ApoE through this mechanism. Recent work suggests that inflammatory astrocytes exert toxic effects via neurotoxic lipids carried by ApoE and another carrier protein, CLU. We find that inhibiting HMGCR in astrocytes does not prevent cytokine‐induced conversion to an inflammatory phenotype, but remains able to block ApoE releaseConclusionWe conclude that targeted inhibition of prenylation enzymes may provide a therapeutic strategy to modulate ApoE levels in the brain.

Statins as Potential Preventative Treatment of ETX and Multiple Pore-Forming Toxin-Induced Diseases.

Epsilon toxin (ETX), produced by type B and D strains of Clostridium perfringens, can cause fatal enterotoxaemia in ruminant animals, particularly sheep, cattle, and goats. Previous studies show that the cytotoxicity of ETX is dependent on the integrity of lipid rafts, the maintenance of which is ensured by cholesterol. Zaragozic acid (ZA) is a statin drug that reduces the synthesis of squalene, which is responsible for cholesterol synthesis. In this study, ZA significantly reduced the toxicity of ETX in Madin-Darby canine kidney (MDCK) cells. We show that ZA does not affect the binding of ETX to MDCK cells, but propidium iodide staining (PI) and Western blotting confirmed that ZA significantly disrupts the ability of ETX to form pores or oligomers in MDCK cells. Additionally, ZA decreased the phosphatidylserine exposure on the plasma membrane and increased the Ca2+ influx of the cells. Results of density gradient centrifugation suggest that ZA decreased the number of lipid rafts in MDCK membranes, which probably contributed to the attenuation of pore-formation. Moreover, ZA protected mice against ETX in vivo. All mice pre-treated with ZA for 48 h before exposure to an absolute lethal dose of ETX (6400 ng/kg) survived. In summary, these findings provide an innovative method to prevent ETX intoxication. Considering many pore-forming toxins require lipid rafts, we tested and found ZA also inhibited the toxicity of other toxins such as Clostridium perfringens Net B and β-toxin (CPB) and Staphylococcus aureus α-hemolysin (Hla). We expect ZA can thus be developed as a broad-spectrum medicine for the treatment of multiple toxins. In addition, other statins, such as lovastatin (LO), also reduced the toxicity of ETX. These findings indicate that statin medicines are potential candidates for preventing and treating multiple toxin-induced diseases.

Establishment of Vero cell lines persistently harboring a yellow fever virus 17D subgenomic replicon

Yellow fever virus (YFV), a member of the genus Flavivirus, family Flaviviridae, is the etiological agent for an acute viral hemorrhagic disease, yellow fever. Although effective live attenuated vaccines based on the strain YFV 17D are currently available, no specific antiviral drug is available, and the disease remains a major public health concern. Hence, the discovery and development of antiviral drugs should lead to great benefits in controlling the disease. To provide a screening platform for antiviral agents targeting YFV RNA translation/replication, we have established and characterized two Vero cell lines that persistently harbor a subgenomic replicon derived from YFV 17D-204 (referred to as replicon cells). The replicon carries YFV nucleotides (1 − 176 and 2382−10,862) and a green fluorescent protein (GFP)-Zeocin resistance fusion gene as a selection marker and indicator of persistent replication. Immunofluorescence analysis revealed that both replicon cells and YFV 17D-infected cells showed similar distribution patterns of viral NS4B protein and replication intermediate, double-stranded RNA. Sequencing analysis of persistent replicons from the two replicon cell lines suggested that their nucleotide sequences did not vary greatly following multiple passages. We examined the effect of five agents, the antiviral cytokines interferon-β and -γ, the nucleoside analog ribavirin, the squalene synthase inhibitor zaragozic acid A, and the antibiotic rifapentine, a recently reported entry and replication inhibitor against YFV, on the persistent replication in the two replicon cell lines. These agents were selected because they inhibited both production of YFV 17D and transient replication of a luciferase-expressing replicon in Vero cells, without greatly affecting cell viability. We found that each of the agents decreased GFP fluorescence in the replicon cells, albeit to varying degrees. The agents other than rifapentine also showed a decrease in viral RNA levels in the replicon cells comparable to that seen for GFP fluorescence. These results indicate that persistent replication is susceptible to each of these five agents, although their mechanisms of action may differ. Taken together, these results provide evidence that translation/replication of the replicon in the replicon cells mimics that of the viral genome upon YFV 17D infection, indicating that the replicon cell lines can serve as a useful tool for high-throughput antiviral drug screening.

Cluster Preface: Organic Photoredox Catalysis in Synthesis – Honoring Professor Shunichi Fukuzumi’s 70th Birthday

Dave Nicewicz was born and raised in the United States in Central New Jersey, before moving to North Carolina, where he completed his Bachelor’s (2000) and Master’s (2001) degrees in Chemistry at the University of North Carolina (USA) at Charlotte with Professor Craig A. Ogle. He then moved to the University of North Carolina at Chapel Hill (USA) where he completed his Ph.D. with Professor Jeffrey S. Johnson. Dave’s studies were focused on the development of acyl anion equivalents generated via 1,2-Brook rearrangements from silylglyoxylates, which he was able to successfully apply to a total synthesis of zaragozic acid C to complete his Ph.D. in 2006. Following his graduate education, Nicewicz moved back to his native New Jersey in 2007, where he was a Ruth L. Kirschstein Postdoctoral Fellow in the laboratories of Professor David W. C. MacMillan. It was during this time that Nicewicz pioneered the use of ruthenium photoredox catalysis in combination with chiral amine organocatalysis to develop a general method for enantioselective aldehyde alkylation. In July of 2009, Dave went on to begin his independent career at the University of North Carolina at Chapel Hill, where his laboratory has focused on organic photoredox catalysis for the development of novel chemical reactivity. He has received a number of awards early on in his career from the University of North Carolina (James Moeser Award for Distinguished Research; Ruth Hettleman Prize for Artistic and Scholarly Achievement), industry (Boehringer Ingelheim New Investigator Award in Organic Chemistry; Amgen Young Investigator Award; Eli Lilly Grantee Award), private foundations (Packard Fellowship in Science and Engineering; Camille Prof. D. A. Nicewicz Dreyfus Teacher-Scholar Award) as well as international recognition (Society of Synthetic Organic Chemistry, Japan Lectureship Award; The 13th Hirata Award, Nagoya University). In 2015, he was promoted to the rank of Associate Professor, where he leads a research group focused on organic methodology development, catalysis and complex molecule synthesis

Toward the stereoselective synthesis of zaragozic acid framework: A desilylation-aldol reaction approach

A convergent synthesis of the C3-C8 fragment of zaragozic acids is described. The key reactions include desilylation-aldol reaction, rearrangement induced by regioselective reductive cleavage, BAIB/TEMPO-Pinnick oxidation, esterification, silylation, and hydrogenolysis.

Age-Dependent Decrease in Hepatic Geranylgeranoic Acid Content in C3H/HeN Mice and Its Oral Supplementation Prevents Spontaneous Hepatoma.

Geranylgeranoic acid (GGA) has been developed as a preventive agent against second primary hepatoma. Recently, GGA was reported to induce cell death in human hepatoma cells via TLR4-mediated pyroptosis. We have reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived cells and that endogenous GGA is found in most organs of rats. In addition, we found that upregulation of endogenous GGA levels by zaragozic acid A (ZAA) induced cell death in human hepatoma-derived cells. Therefore, we investigated the age-related changes in hepatic GGA and the possibility of suppressing hepatocarcinogenesis by GGA supplementation using male C3H/HeN mice that spontaneously develop hepatoma. We measured endogenous GGA and mRNA of monoamine oxidase (BMAOB), a key enzyme of GGA biosynthesis, in the liver of male C3H/HeN mice aged 6–93 weeks. We also tried suppressing spontaneous hepatocarcinogenesis by a single administration of GGA to C3H/HeN mice. Hepatic GGA content and Maob mRNA expression level age-dependently decreased in male C3H/HeN mice; some of which produced spontaneous hepatoma in 2 years. A single oral administration of GGA at 11 months of age significantly prevented hepatoma in terms of the number and weight of tumors per mouse at 24 months. Oral supplementation with GGA or geranylgeraniol significantly increased endogenous hepatic GGA contents dose-dependently; and ZAA dramatically upregulated hepatic GGA. In this study; we found an age-dependent decrease in hepatic endogenous GGA in male C3H/HeN mice and efficient prevention of spontaneous hepatoma by a single administration of GGA at 11 months of age.

Антибиопленочная активность антиматриксных молекул

Внеклеточный матрикс биопленок обеспечивает фиксацию биопленки на биологической поверхности и защиту ее бактерий от внешних неблагоприятных факторов. Основным структурным компонентом био­пленок является внеклеточное полисахаридное вещество. Ключевыми молекулярными структурами, поддерживающими трехмерную структуру биопленки, считают экзополисахариды и амилоидоподобные волокна. До недавнего времени предполагалось, что большинство механизмов диспергирования биопленок ассоциировано с функционированием матриксных деградирующих ферментов, таких как гликозидгидролазы, полисахаридные лиазы и протеазы. Однако продемонстрировано, что в процессе разрушения матриксных экзополисахаридов и амилоидоподобных волокон играют самостоятельную роль малые молекулы. Среди соединений, нарушающих матрикс биопленки, различают антиматриксные молекулы, соединения, взаимодействующие с микродоменами бактериальной мембраны, и бактериальные поверхностно-активные вещества (биосурфагенты). Продемонстрировано, что соединения данных групп могут ингибировать образование биопленок и способствовать их диспергированию. Из группы антиматриксных молекул полиаминное соединение норспермидин взаимодействует с экзополисахаридами, а производные бензохинона AA-861 и сесквитерпеновый лактон партенолид — с TasA-амилоидоподобными волокнами. Норспермидин предотвращает образование и диспергирует биопленки различных бактерий, включая Acinetobacter baumannii, Bacillus subtili, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis. Соединение AA-861 проявляет активность против биопленок, которые образованы бактериями Streptococcus mutans, Bacillus cereus, Escherichia coli, а партенолид диспергирует биопленки, сформированные Escherichia coli и Bacillus cereus. Сарагосовые кислоты, взаимодействуя с микродоменами бактериальной мембраны, нарушают функционирование рафт-ассоциированных протеинов бактерий. Малые антиматриксные молекулы и нацеленные на микродомены мембраны бактерий, инициирующие диспергирование биопленки, безусловно, станут основанием для разработки эффективных антибиопленочных терапевтических лекарственных средств.

Site‐ and Enantioselective Iridium‐Catalyzed Desymmetric Mono‐Hydrogenation of 1,4‐Dienes

AbstractThe control of site selectivity in asymmetric mono‐hydrogenation of dienes or polyenes remains largely underdeveloped. Herein, we present a highly efficient desymmetrization of 1,4‐dienes via iridium‐catalyzed site‐ and enantioselective hydrogenation. This methodology demonstrates the first iridium‐catalyzed hydrogenative desymmetriation of meso dienes and provides a concise approach to the installation of two vicinal stereogenic centers adjacent to an alkene. High isolated yields (up to 96 %) and excellent diastereo‐ and enantioselectivities (up to 99:1 d.r. and 99 % ee) were obtained for a series of divinyl carbinol and divinyl carbinamide substrates. DFT calculations reveal that an interaction between the hydroxy oxygen and the reacting hydride is responsible for the stereoselectivity of the desymmetrization of the divinyl carbinol. Based on the calculated energy profiles, a model that simulates product distribution over time was applied to show an intuitive kinetics of this process. The usefulness of the methodology was demonstrated by the synthesis of the key intermediates of natural products zaragozic acid A and (+)‐invictolide.

Site- and Enantioselective Iridium-Catalyzed Desymmetric Mono-Hydrogenation of 1,4-Dienes.

The control of site selectivity in asymmetric mono‐hydrogenation of dienes or polyenes remains largely underdeveloped. Herein, we present a highly efficient desymmetrization of 1,4‐dienes via iridium‐catalyzed site‐ and enantioselective hydrogenation. This methodology demonstrates the first iridium‐catalyzed hydrogenative desymmetriation of meso dienes and provides a concise approach to the installation of two vicinal stereogenic centers adjacent to an alkene. High isolated yields (up to 96 %) and excellent diastereo‐ and enantioselectivities (up to 99:1 d.r. and 99 % ee) were obtained for a series of divinyl carbinol and divinyl carbinamide substrates. DFT calculations reveal that an interaction between the hydroxy oxygen and the reacting hydride is responsible for the stereoselectivity of the desymmetrization of the divinyl carbinol. Based on the calculated energy profiles, a model that simulates product distribution over time was applied to show an intuitive kinetics of this process. The usefulness of the methodology was demonstrated by the synthesis of the key intermediates of natural products zaragozic acid A and (+)‐invictolide.

Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis

ABSTRACT Rotavirus is the most common cause of severe diarrhea among infants and young children and is responsible for more than 200,000 pediatric deaths per year. There is currently no pharmacological treatment for rotavirus infection in clinical activity. Although cholesterol synthesis has been proven to play a key role in the infections of multiple viruses, little is known about the relationship between cholesterol biosynthesis and rotavirus replication. The models of rotavirus infected two cell lines and a human small intestinal organoid were used. We investigated the effects of cholesterol biosynthesis, including inhibition, enhancement, and their combinations on rotavirus replication on these models. The knockdown of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) was built by small hairpin RNAs in Caco2 cells. In all these models, inhibition of cholesterol synthesis by statins or HMGCR knockdown had a significant inhibitory effect on rotavirus replication. The result was further confirmed by the other inhibitors: 6-fluoromevalonate, Zaragozic acid A and U18666A, in the cholesterol biosynthesis pathway. Conversely, enhancement of cholesterol production increased rotavirus replication, suggesting that cholesterol homeostasis is relevant for rotavirus replication. The effects of all these compounds toward rotavirus were further confirmed with a clinical rotavirus isolate. We concluded that rotavirus replication is dependent on cholesterol biosynthesis. To be specific, inhibition of cholesterol synthesis can downregulate rotavirus replication; on the contrary, rotavirus replication is upregulated. Statin treatment is potentially an effective novel clinical anti-rotavirus strategy.

The effect of bile acids on the growth and global gene expression profiles in Akkermansia muciniphila

Akkermansia muciniphila is a prominent member of the gut microbiota and the organism gets exposed to bile acids within this niche. Several gut bacteria have bile response genes to metabolize bile acids or an ability to change their membrane structure to prevent membrane damage from bile acids. To understand the response to bile acids and how A. muciniphila can persist in the gut, we studied the effect of bile acids and individual bile salts on growth. In addition, the change in gene expression under ox-bile condition was studied. The growth of A. muciniphila was inhibited by ox-bile and the bile salts mixture. Individual bile salts have differential effects on the growth. Although most bile salts inhibited the growth of A. muciniphila, an increased growth was observed under culture conditions with sodium deoxycholate. Zaragozic acid A, which is a squalene synthase inhibitor leading to changes in the membrane structure, increased the susceptibility of A. muciniphila to bile acids. Transcriptome analysis showed that gene clusters associated with an ABC transporter and RND transporter were upregulated in the presence of ox-bile. In contrast, a gene cluster containing a potassium transporter was downregulated. Membrane transporter inhibitors also decreased the tolerance to bile acids of A. muciniphila. Our results indicated that membrane transporters and the squalene-associated membrane structure could be major bile response systems required for bile tolerance in A. muciniphila.Key points• The growth of Akkermansia muciniphila was inhibited by most bile salts.• Sodium deoxycholate increased the growth of A. muciniphila.• The genes encoding transporters and hopanoid synthesis were upregulated by ox-bile.• The inhibitors of transporters and hopanoid synthesis reduced ox-bile tolerance.

Squalene lipotoxicity in a lipid droplet-less yeast mutant is linked to plasma membrane dysfunction.

Squalene is a naturally occurring triterpene with wide industrial applications. Due to limited natural resources, production of this valuable lipid in yeast is of high commercial relevance. Typically low levels of squalene in yeast can be significantly increased by specific cultivation conditions or genetic modifications. Under normal conditions, excess squalene is stored in lipid droplets (LD), while in a Saccharomyces cerevisiae mutant unable to form LD it is distributed to cellular membranes. We present here the evidence that squalene accumulation in this LD-less mutant treated with squalene monooxygenase inhibitor terbinafine induces growth defects and loss of viability. We show that plasma membrane malfunction is involved in squalene toxicity. We have found that subinhibitory concentrations of terbinafine increased the sensitivity of LD-less mutant to several membrane-active substances. Furthermore, squalene accumulation in terbinafine-treated LD-less cells disturbed the maintenance of membrane potential and increased plasma membrane permeability to rhodamine 6G. LD-less cells treated with terbinafine showed also high sensitivity to osmotic stress. To confirm the causal relationship between squalene accumulation, loss of viability and impaired plasma membrane functions we treated LD-less cells simultaneously with terbinafine and squalene synthase inhibitor zaragozic acid. Reduction of squalene levels by zaragozic acid improved cell growth and viability and decreased plasma membrane permeability to rhodamine 6G in terbinafine-treated LD-less cells. Our results support the hypothesis that plasma membrane malfunction is involved in the mechanisms of squalene lipotoxicity in yeast cells with defective lipid storage.

Development of Synthetic Strategies for Densely Oxygenated Natural Products: Total Synthesis of Lactacystin and Zaragozic Acid C Using Photochemical C(sp3)-H Functionalization

This review describes two novel synthetic routes from (S)-pyroglutaminol to (+)-lactacystin, a potent inhibitor of the 20S proteasome and from d-gluconolactone derivative to zaragozic acid C, a potent squalene synthase inhibitor. In lactacystin synthesis, the photoinduced intermolecular C(sp3)-H alkynylation and intramolecular C(sp3)-H acylation chemoselectively and stereoselectively constructed the tetrasubstituted and trisubstituted carbon centers, respectively. In the synthesis of zaragozic acid C, the stereoselective installation of the two contiguous tetrasubstituted carbons was achieved by the photochemical intramolecular C(sp3)-H acylation of a densely oxygenated intermediate.

Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production. Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication.

Aspergillus flavus squalene synthase as an antifungal target: Expression, activity, and inhibition

Invasive aspergillosis (IA) is a life-threatening disease impacting immunocompromised individuals. Standard treatments of IA, including polyenes and azoles, suffer from high toxicity and emerging resistance, leading to the need to develop new antifungal agents with novel mechanisms of action. Ergosterol biosynthesis is a classic target for antifungals, and squalene synthase (SQS) catalyzes the first committed step in ergosterol biosynthesis in Aspergillus spp. making SQS of interest in the context of antifungal development. Here, we cloned, expressed, purified and characterized SQS from the pathogen Aspergillus flavus (AfSQS), confirming that it produced squalene. To identify potential leads targeting AfSQS, we tested known squalene synthase inhibitors, zaragozic acid and the phosphonosulfonate BPH-652, finding that they were potent inhibitors. We then screened a library of 744 compounds from the National Cancer Institute (NCI) Diversity Set V for inhibition activity. 20 hits were identified and IC50 values were determined using dose-response curves. 14 compounds that interfered with the assay were excluded and the remaining 6 compounds were analyzed for drug-likeness, resulting in one compound, celastrol, which had an AfSQS IC50 value of 830 nM. Enzyme inhibition kinetics revealed that celastrol binds to AfSQS in a noncompetitive manner, but did not bind covalently. Since celastrol is also known to inhibit growth of the highly virulent Aspergillus fumigatus by inhibiting flavin-dependent monooxygenase siderophore A (SidA, under iron starvation conditions), it may be a promising multi-target lead for antifungal development.

Unequivocal evidence for endogenous geranylgeranoic acid biosynthesized from mevalonate in mammalian cells

Geranylgeranoic acid (GGA) has been reported to induce autophagic cell death via upregulation of lipid-induced unfolded protein response in several human hepatoma-derived cell lines, and its 4,5-didehydro derivative has been developed as a preventive agent against second primary hepatoma in clinical trials. We have previously reported that GGA is a natural diterpenoid synthesized in several medicinal herbs. Here, we provide unequivocal evidence for de novo GGA biosynthesis in mammals. First, with normal male Wistar rats, the levels of GGA in liver were found to be far greater than those in other organs analyzed. Second, we demonstrated the metabolic GGA labeling from the 13C-labeled mevalonolactone in the human hepatoma-derived cell line, HuH-7. Isotopomer spectral analysis revealed that approximately 80% of the cellular GGA was newly synthesized from mevalonate (MVA) in 12 h and the acid picked up preexisting farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), suggesting that GGA is derived from FPP and GGPP through the MVA pathway. Third, zaragozic acid A, a squalene synthase inhibitor, induced dose-dependent upregulation of endogenous GGA content in HuH-7 cells and their concomitant cell death. These results strongly suggest that a cancer-preventive GGA is biosynthesized via the MVA pathway in mammals.

Repositioning of Tak-475 In Mevalonate Kinase Disease: Translating Theory Into Practice.

Mevalonate Kinase Deficiency (MKD, OMIM #610377) is a rare autosomal recessive metabolic and inflammatory disease. In MKD, defective function of the enzyme mevalonate kinase, due to a mutation in the MVK gene, leads to the shortage of mevalonate- derived intermediates, which results in unbalanced prenylation of proteins and altered metabolism of sterols. These defects lead to a complex multisystem inflammatory and metabolic syndrome. Although biologic therapies aimed at blocking the inflammatory cytokine interleukin- 1 can significantly reduce inflammation, they cannot completely control the clinical symptoms that affect the nervous system. For this reason, MKD can still be considered an orphan drug disease. The availability of MKD models reproducing the MKD-systematic inflammation, is crucial to improve the knowledge on its pathogenesis, which is still unknown. New therapies are also required in order to improve pateints' conditions and their quality of life. MKD-cellular models can be obtained by biochemical inhibition of mevalonatederived isoprenoids. Of note, these cells present an exaggerated response to inflammatory stimuli that can be reduced by treatment with zaragozic acid, an inhibitor of squalene synthase, thus increasing the availability of isoprenoids intermediates upstream the enzymatic block. A similar action might be obtained by lapaquistat acetate (TAK-475, Takeda), a drug that underwent extensive clinical trials as a cholesterol lowering agent 10 years ago, with a good safety profile. Here we describe the preclinical evidence supporting the possible repositioning of TAK-475 from its originally intended use to the treatment of MKD and discuss its potential to modulate the mevalonate pathway in inflammatory diseases.

Helicobacter pylori Depletes Cholesterol in Gastric Glands to Prevent Interferon Gamma Signaling and Escape the Inflammatory Response

Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosafor decades. H pylori expression of cholesterol-α-glucosyltransferase(encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response. MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patientsundergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-β-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed byimmunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Δcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1-/- mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Δcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy. In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human β-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNG-response gene IRF1 was substantially higher in PMSS1Δcgt-infected mice than PMSS1-infected mice. Ifngr1-/- mice were colonized by PMSS1 to a greater extent than control mice. H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which Hpylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell-based vaccines against H pylori.

Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin

Animal health depends on the ability of immune cells to kill invading pathogens, and on the resilience of tissues to tolerate the presence of pathogens. Trueperella pyogenes causes tissue pathology in many mammals by secreting a cholesterol-dependent cytolysin, pyolysin (PLO), which targets stromal cells. Cellular cholesterol is derived from squalene, which is synthesized via the mevalonate pathway enzymes, including HMGCR, FDPS and FDFT1. The present study tested the hypothesis that inhibiting enzymes in the mevalonate pathway to reduce cellular cholesterol increases the resilience of stromal cells to PLO. We first verified that depleting cellular cholesterol with methyl-β-cyclodextrin increased the resilience of stromal cells to PLO. We then used siRNA to deplete mevalonate pathway enzyme gene expression, and used pharmaceutical inhibitors, atorvastatin, alendronate or zaragozic acid to inhibit the activity of HMGCR, FDPS and FDFT1, respectively. These approaches successfully reduced cellular cholesterol abundance, but mevalonate pathway enzymes did not affect cellular resilience equally. Inhibiting FDFT1 was most effective, with zaragozic acid reducing the impact of PLO on cell viability. The present study provides evidence that inhibiting FDFT1 increases stromal cell resilience to a cholesterol-dependent cytolysin.