Overexpression and Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA Synthase Affect Central Metabolic Pathways in Tobacco.

Little has been established on the relationship between the mevalonate (MVA) pathway and other metabolic pathways except for the sterol and glucosinolate biosynthesis pathways. In the MVA pathway, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to form 3-hydroxy-3-methylglutaryl-coenzyme A. Our previous studies had shown that, while the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited higher sterol content, growth rate and seed yield than OE-wtBjHMGS1. Herein, untargeted proteomics and targeted metabolomics were employed to understand the phenotypic effects of HMGS overexpression in tobacco by examining which other metabolic pathways were affected. Sequential window acquisition of all theoretical mass spectra quantitative proteomics analysis on OE-wtBjHMGS1 and OE-S359A identified the misregulation of proteins in primary metabolism and cell wall modification, while some proteins related to photosynthesis and the tricarboxylic acid cycle were upregulated in OE-S359A. Metabolomic analysis indicated corresponding changes in carbohydrate, amino acid and fatty acid contents in HMGS-OEs, and F-244, a specific inhibitor of HMGS, was applied successfully on tobacco to confirm these observations. Finally, the crystal structure of acetyl-CoA-liganded S359A revealed that improved activity of S359A likely resulted from a loss in hydrogen bonding between Ser359 and acyl-CoA, which is evident in wtBjHMGS1. This work suggests that regulation of plant growth by HMGS can influence the central metabolic pathways. Furthermore, this study demonstrates that the application of the HMGS-specific inhibitor (F-244) in tobacco represents an effective approach for studying the HMGS/MVA pathway.

Silencing of peroxisome proliferator-activated receptor-alpha alleviates myocardial injury in diabetic cardiomyopathy by downregulating 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 expression.

Diabetic cardiomyopathy (DCM) is a cardiac disorder, which affects around 12% diabetic patients, resulting in overt heart death. Our initial bioinformatic analysis identified the differentially expressed gene 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 (HMGCS2) in DCM, which may be activated by peroxisome proliferator-activated receptor-alpha (PPARα) based on previous evidence. Therefore, the present study aims to explore the effect of PPARα on the development of DCM through regulating HMGCS2. The expression of PPARα and HMGCS2 was detected by reverse transcription quantitative polymerase chain reaction in cardiomyocytes and high-glucose-cultured cardiomyocytes. The proliferation and apoptosis of cardiomyocytes were examined by 5-ethynyl-2'-deoxyuridine assay and flow cytometry, separately. Mitoehondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) levels were determined. Then, the protein levels of B-cell lymphoma 2, Bcl-2-associated X protein, and cleaved Caspase-3 were detected by Western blot analysis. The myocardial apoptosis index, heart weight, and serum lipids of rats were examined. At last, the expressions of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), transforming growth factor ß1 (TGFß1), peroxisome proliferator activator receptor gamma coactivator-1 alpha (PGC1α), nuclear respiratory factor (NRF)-1, NRF-2, NAD(P)H oxidase 1, and superoxide dismutase-1 were examined. HMGCS2 was the most differentially expressed gene in DCM. The levels of HMGCS2 and PPARα were upregulated in patients with DCM. HMGCS2 silencing was shown to inhibit HMGCS2 expression to suppress the apoptosis of high-glucose-induced cardiomyocytes and the loss of MMP, reduce the accumulation of ROS, and promote cardiomyocyte proliferation. Silencing of HMGCS2 and PPARα alleviated myocardial injury, decreased blood glucose, and lipid in DCM rats, downregulated the expression of ANP, BNP, and TGFß1 to reduce myocardial injury, and elevated PGC1α, NRF-1, and NRF-2 levels to enhance oxidative stress levels. Our results demonstrated that silencing of PPARα could alleviate cardiomyocyte injury and oxidative stress via a mechanism related to the downregulation of HMGCS2, which could provide a novel target for DCM treatment.

HMG-CoA synthase 1 is a synthetic lethal partner of BRAFV600E in human cancers.

Contributions of metabolic changes to cancer development and maintenance have received increasing attention in recent years. Although many human cancers share similar metabolic alterations, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Using an RNAi-based screen targeting the majority of the known metabolic proteins, we recently found that oncogenic BRAFV600E up-regulates HMG-CoA lyase (HMGCL), which converts HMG-CoA to acetyl-CoA and a ketone body, acetoacetate, that selectively enhances BRAFV600E-dependent MEK1 activation in human cancer. Here, we identified HMG-CoA synthase 1 (HMGCS1), the upstream ketogenic enzyme of HMGCL, as an additional "synthetic lethal" partner of BRAFV600E Although HMGCS1 expression did not correlate with BRAFV600E mutation in human melanoma cells, HMGCS1 was selectively important for proliferation of BRAFV600E-positive melanoma and colon cancer cells but not control cells harboring active N/KRAS mutants, and stable knockdown of HMGCS1 only attenuated colony formation and tumor growth potential of BRAFV600E melanoma cells. Moreover, cytosolic HMGCS1 that co-localized with HMGCL and BRAFV600E was more important than the mitochondrial HMGCS2 isoform in BRAFV600E-expressing cancer cells in terms of acetoacetate production. Interestingly, HMGCL knockdown did not affect HMGCS1 expression levels, whereas HMGCS1 knockdown caused a compensating increase in HMGCL protein level because of attenuated protein degradation. However, this increase did not reverse the reduced ketogenesis in HMGCS1 knockdown cells. Mechanistically, HMGCS1 inhibition decreased intracellular acetoacetate levels, leading to reduced BRAFV600E-MEK1 binding and consequent MEK1 activation. We conclude that the ketogenic HMGCS1-HMGCL-acetoacetate axis may represent a promising therapeutic target for managing BRAFV600E-positive human cancers.

HMGCS2 promotes autophagic degradation of the amyloid-ß precursor protein through ketone body-mediated mechanisms.

HMGCS2 (mitochondrial 3-hydroxy-3-methylglutaryl-COA synthase 2) is a control enzyme in ketogenesis. The mitochondrial localization and interaction with APP (ß-amyloid precursor protein) suggest that HMGCS2 may play a role in the pathophysiology of AD (Alzheimer's disease). Here we report that overexpression of HMGCS2 decreased levels of APP and related CTFs (carboxy-terminal fragments), which was largely prevented by an autophagic inhibitor chloroquine. In addition, HMGCS2 enhancement of autophagic marker LC3II was diminished by rapamycin, an inhibitor of mechanistic target of rapamycin. Moreover, deprivation of EBSS (Earle's Balanced Salt Solution) significantly augmented the effect of HMGCS2 on LC3II, while acetoacetate reversed the reduction of LC3II, APP and CTFs which was induced by HMGCS2 knockdown. In the presence of acetoacetate, rapamycin failed to induce further increase of LC3II, which mimicked the effect of HMGCS2 overexpression. Finally, HMGCS2 enhanced the antioxidant response. Collectively, HMGCS2 shares with ketone bodies common features in autophagic clearance of APP and CTFs, suggesting that ketone bodies play an important role in HMGCS2 regulation of the autophagy.

Momilactone B Inhibits Ketosis In Vitro by Regulating the ANGPTL3-LPL Pathway and Inhibiting HMGCS2.

Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.

PPARα-dependent cholesterol/testosterone disruption in Leydig cells mediates 2,4-dichlorophenoxyacetic acid-induced testicular toxicity in mice.

It was reported that 2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide and a possible endocrine disruptor, can disturb spermatogenesis, but the precise mechanism is not understood. Since 2,4-D is a weak peroxisome proliferator in hepatocytes and peroxisome proliferator-activated receptor α (PPARα) is also expressed in Leydig cells, this study aimed to investigate the link between PPARα and 2,4-D-mediated testicular dysfunction. 2,4-D (130 mg/kg/day) was administered to wild-type and Ppara-null mice for 2 weeks, and the alterations in testis and testosterone/cholesterol metabolism in Leydig cells were examined. Treatment with 2,4-D markedly decreased testicular testosterone in wild-type mice, leading to degeneration of spermatocytes and Sertoli cells. The 2,4-D decreased cholesterol levels in Leydig cells of wild-type mice through down-regulating the expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 and reductase, involved in de novo cholesterogenesis. However, the mRNAs encoding the important proteins involved in testosterone synthesis were unchanged by 2,4-D except for CYP17A1, indicating that exhausted cholesterol levels in the cells is a main reason for reduced testicular testosterone. Additionally, pregnancy rate and the number of pups between 2,4-D-treated wild-type male mice and untreated female mice were significantly lower compared with those between untreated couples. These phenomena were not observed in 2,4-D-treated Ppara-null males. Collectively, these results suggest a critical role for PPARα in 2,4-D-induced testicular toxicity due to disruption of cholesterol/testosterone homeostasis in Leydig cells. This study yields novel insights into the possible mechanism of testicular dysfunction and male infertility caused by 2,4-D.

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin.

Hymeglusin (1233A, F244, L-659-699) is established as a specific ß-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 Å) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.

Ceestatin, a novel small molecule inhibitor of hepatitis C virus replication, inhibits 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

BACKGROUND: Hepatitis C virus (HCV) chronically infects >170 million persons worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. The identification of more effective and better-tolerated agents for treating HCV is a high priority. We have reported elsewhere the discovery of the anti-HCV compound ceestatin using a high-throughput screen of a small molecule library. METHODS: To identify host or viral protein targets in an unbiased fashion, we performed affinity chromatography, using tandem liquid chromatography/mass spectrometry to identify specific potential targets. RESULTS. Ceestatin binds to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and irreversibly inhibits HMG-CoA synthase in a dose-dependent manner. Ceestatin's anti-HCV effects are reversed by addition of HMG-CoA, mevalonic acid, or geranylgeraniol. Treatment with small interfering RNA against HMG-CoA synthase led to a substantial reduction in HCV replication, further validating HMG-CoA synthase as an enzyme essential for HCV replication. CONCLUSIONS: Ceestatin therefore exerts its anti-HCV effects through inhibition of HMG-CoA synthase. It may prove useful as an antiviral agent, as a probe to study HCV replication, and as a cholesterol-lowering agent. The logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to uncover the targets on which novel uncharacterized anti-HCV compounds act.

HMGCS1 drives drug-resistance in acute myeloid leukemia through endoplasmic reticulum-UPR-mitochondria axis.

Hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) is a key enzyme in the mevalonate pathway of cholesterol synthesis. Dysregulation of HMGCS1 expression is a common occurrence in many solid tumors. It was also found to be overexpressed in newly diagnosed (ND) and relapsed/refractory (RR) acute myeloid leukemia (AML) patients. Previous study proved that HMGCS1 could induce drug-resistance in AML cells. However, the underlying mechanism how HMGCS1 contributed to chemoresistance remains elusive. Here, we confirmed that HMGCS1 inhibitor Hymeglusin enhanced cytarabine/Adriamycin (Ara-c/ADR) chemo-sensitivity in AML cells lines. Moreover, Ara-c-resistant HL-60 cells (HL-60/Ara-c) and ADR-resistant HL-60 cells (HL-60/ADR) were more sensitive to HMGCS1 inhibition than HL-60 cells. In addition, we demonstrated that the transcription factor GATA1 was the upstream regulator of HMGCS1 and could directly bind to the HMGCS1 promoter. After treatment of Tunicamycin (Tm), the number of mitochondria was increased and the damage of endoplasmic reticulum (ER) was reduced in bone marrow cells from AML-RR patients, compared to cells from AML-CR group. HMGCS1 protected mitochondria and ER under ER stress and up-regulated unfold protein response (UPR) downstream molecules in AML cells. In summary, we proved that HMGCS1 could upregulate UPR downstream components, protect mitochondria and ER from damage in AML cells under stress, therefore conferring drug resistance. Therefore, HMGCS1 could serve as a novel target for treatment of patients with intolerant chemotherapy and AML-RR patients.

A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase catalyzes the first physiologically irreversible step in biosynthesis of isoprenoids and sterols from acetyl-CoA. Inhibition of enzyme activity by beta-lactone-containing natural products correlates with substantial diminution of sterol synthesis, identifying HMG-CoA synthase as a potential drug target and suggesting that identification of effective inhibitors would be valuable. A visible wavelength spectrophotometric assay for HMG-CoA synthase has been developed. The assay uses dithiobisnitrobenzoic acid (DTNB) to detect coenzyme A (CoASH) release on acetylation of enzyme by the substrate acetyl-CoA, which precedes condensation with acetoacetyl-CoA to form the HMG-CoA product. The assay method takes advantage of the stability of recombinant enzyme in the absence of a reducing agent. It can be scaled down to a 60 microl volume to allow the use of 384-well microplates, facilitating high-throughput screening of compound libraries. Enzyme activity measured in the microplate assay is comparable to values measured by using conventional scale spectrophotometric assays with the DTNB method (412 nm) for CoASH production or by monitoring the use of a second substrate, acetoacetyl-CoA (300 nm). The high-throughput assay method has been successfully used to screen a library of more than 100,000 drug-like compounds and has identified both reversible and irreversible inhibitors of the human enzyme.

Induction of secondary metabolite production by hygromycin B and identification of the 1233A biosynthetic gene cluster with a self-resistance gene.

We found that the protein synthesis inhibitor hygromycin B induced the production of secondary metabolites, including lucilactaene, NG-391, fusarubin, 1233A, and 1233B, in the filamentous fungus, Fusarium sp. RK97-94. We identified the biosynthetic gene cluster for 1233A, an HMG-CoA synthase inhibitor. The biosynthetic gene cluster consisted of four genes, one of which was involved in conferring self-resistance to 1233A.

HMG-CoA synthase 2 drives brain metabolic reprogramming in cocaine exposure.

The brain is a high energy-consuming organ that typically utilizes glucose as the main energy source for cerebral activity. When glucose becomes scarce under conditions of stress, ketone bodies, such as ß-hydroxybutyrate, acetoacetate and acetone, become extremely important. Alterations in brain energy metabolism have been observed in psychostimulant abusers; however, the mode of brain metabolic programming in cocaine dependence remains largely unknown. Here, we profiled the metabolites and metabolic enzymes from brain nucleus accumbens (NAc) of mice exposed to cocaine. We found that cocaine modified energy metabolism and markedly activated ketogenesis pathway in the NAc. The expression of HMG-CoA synthase 2 (HMGCS2), a critical rate-limiting ketogenesis enzyme, was markedly up-regulated. After switching metabolic pathways from ketogenesis to glycolysis through activation of glucokinase, cocaine-evoked metabolic reprogramming regained homeostasis, and the cocaine effect was attenuated. Importantly, both the pharmacological and genetic inhibition of HMGCS2 significantly suppressed cocaine-induced ketogenesis and behavior. In conclusion, cocaine induces a remarkable energy reprogramming in the NAc, which is characterized by HMGCS2-driven ketogenesis. Such effect may facilitate adaptations to cocaine-induced energy stress in the brain. Our findings establish an important link between drug-induced energy reprogramming and cocaine effect, and may have implication in the treatment of cocaine addiction.

Inactivation of the Ras/MAPK/PPARγ signaling axis alleviates diabetic mellitus-induced erectile dysfunction through suppression of corpus cavernosal endothelial cell apoptosis by inhibiting HMGCS2 expression.

PURPOSE: Diabetic mellitus-induced erectile dysfunction (DMED) represents a significant complication associated with diabetes mellitus (DM) that greatly affects human life quality. Various reports have highlighted the involvement of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) in the regulation of mitochondrial fatty acid oxidation, which has also been linked with DM. Through bioinformatics analysis, HMGCS2 was determined to be a novel target among DM patients suffering from erectile dysfunction (ED), and enriched in the Ras/ERK/PPAR signaling axis. Owing to the fact that the key mechanism HMGCS2 involved in DM remains largely unknown, we set out to investigate the role of the Ras/MAPK/PPARγ signaling axis and HMGCS2 in the corpus cavernosal endothelial cells (CCECs) of rats with DMED. METHODS: Firstly, bioinformatics analysis was used to screen out differentially expressed genes in DMED. Then, to investigate the influence of the Ras/MAPK/PPARγ signaling axis and HMGCS2 on DMED, a rat model of DMED was established and injected with Simvastatin and si-Hmgcs2. The individual expression patterns of Ras, MAPK, PPARγ and HMGCS2 were determined by RT-qPCR, immunohistochemistry and western blot analysis methods. Afterwards, to investigate the mechanism of Ras/MAPK/PPARγ signaling axis and HMGCS2, CCECs were isolated from DMED rats and transfected with agonists and inhibitors of the Ras/MAPK/PPARγ signaling axis and siRNA of HMGCS2, with their respective functions in apoptosis and impairment of CCECs evaluated using TUNEL staining and flow cytometry. RESULTS: Microarray analysis and KEGG pathway enrichment analysis revealed that Ras/ERK/PPAR signaling axis mediated HMGCS2 in DMED. Among the DMED rats, the Ras/MAPK/PPAR signaling axis was also activated while the expression of HMGCS2 was upregulated. The activation of Ras was determined to be capable of upregulating ERK expression which resulted in the inhibition of the transcription of PPARγ and subsequent upregulation of HMGCS2 expression. The inhibited activation of the Ras/ERK/PPAR signaling axis and silencing HMGCS2 were observed to provide an alleviatory effect on the injury of DMED while acting to inhibit the apoptosis of CCECs. CONCLUSION: Collectively, the key findings suggested that suppression of the Ras/MAPK/PPARγ signaling axis could downregulate expression of HMGCS2, so as to alleviate DMED. This study defines the potential treatment for DMED through inhibition of the Ras/MAPK/PPARγ signaling axis and silencing HMGCS2.

The nuclear receptor-coactivator interaction surface as a target for peptide antagonists of the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARalpha, PPARdelta, and PPARgamma) constitute a family of nuclear receptors that regulates metabolic processes involved in lipid and glucose homeostasis. Although generally considered to function as ligand-regulated receptors, all three PPARs exhibit a high level of constitutive activity that may result from their stimulation by intracellularly produced endogenous ligands. Consequently, complete inhibition of PPAR signaling requires the development of inverse agonists. However, the currently available small molecule antagonists for the PPARs function only as partial agonists, or their efficacy is not sufficient to inhibit the constitutive activity of these receptors. Due to the lack of efficacious antagonists that interact with the ligand-binding domain of the PPARs, we decided to target an interaction that is central to nuclear receptor-mediated gene transcription: the nuclear receptor-coactivator interaction. We utilized phage display technology to identify short LXXLL-containing peptides that bind to the PPARs. Analysis of these peptides revealed a consensus binding motif consisting of HPLLXXLL. Cross-screening of these peptides for binding to other nuclear receptors enabled the identification of a high-affinity PPAR-selective peptide that has the ability to repress PPARgamma1-dependent transcription of transfected reporter genes. Most importantly, when introduced into HepG2 cells, the peptide inhibited the expression of endogenous PPARgamma1 target genes, adipose differentiation-related protein and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase 2. This work lends support for the rational development of peptidomimetics that block receptor-mediated transcription by targeting the nuclear receptor-coactivator interaction surface.

Ketogenesis contributes to intestinal cell differentiation.

The intestinal epithelium undergoes a continual process of proliferation, differentiation and apoptosis. Previously, we have shown that the PI3K/Akt/mTOR pathway has a critical role in intestinal homeostasis. However, the downstream targets mediating the effects of mTOR in intestinal cells are not known. Here, we show that the ketone body ß-hydroxybutyrate (ßHB), an endogenous inhibitor of histone deacetylases (HDACs) induces intestinal cell differentiation as noted by the increased expression of differentiation markers (Mucin2 (MUC2), lysozyme, IAP, sucrase-isomaltase, KRT20, villin, Caudal-related homeobox transcription factor 2 (CDX2) and p21Waf1). Conversely, knockdown of the ketogenic mitochondrial enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) attenuated spontaneous differentiation in the human colon cancer cell line Caco-2. Overexpression of HMGCS2, which we found is localized specifically in the more differentiated portions of the intestinal mucosa, increased the expression of CDX2, thus further suggesting the contributory role of HMGCS2 in intestinal differentiation. In addition, mice fed a ketogenic diet demonstrated increased differentiation of intestinal cells as noted by an increase in the enterocyte, goblet and Paneth cell lineages. Moreover, we showed that either knockdown of mTOR or inhibition of mTORC1 with rapamycin increases the expression of HMGCS2 in intestinal cells in vitro and in vivo, suggesting a possible cross-talk between mTOR and HMGCS2/ßHB signaling in intestinal cells. In contrast, treatment of intestinal cells with ßHB or feeding mice with a ketogenic diet inhibits mTOR signaling in intestinal cells. Together, we provide evidence showing that HMGCS2/ßHB contributes to intestinal cell differentiation. Our results suggest that mTOR acts cooperatively with HMGCS2/ßHB to maintain intestinal homeostasis.

F-244 specifically inhibits 3-hydroxy-3-methylglutaryl coenzyme A synthase.

A beta-lactone isolated from Scopulariopsis sp. shows a potent inhibition of cholesterogenesis. The structure of this beta-lactone, termed F-244, is 3,5,7-trimethyl-12-hydroxy-13-hydroxymethyl-2,4-tetradecadiendioic acid 12,14-lactone. The inhibition site of F-244 in cholesterol synthesis was studied. The growth of Vero cells was inhibited at 6.25-12.5 micrograms/ml of F-244. The inhibition of growth was overcome by the addition of mevalonate to the culture medium, but not by the addition of acetate. In a rat liver enzyme system, the incorporations of [14C]acetate and [14C]acetyl-CoA into digitonin-precipitable sterol were 50% inhibited by 0.58 microgram/ml of F-244. The incorporation of [14C]mevalonate was not affected. Studies on the effects of F-244 on the three enzymes involved in mevalonate biosynthesis demonstrated that the drug specifically inhibits HMG-CoA synthase with IC50 value of 0.065 microgram/ml. The effect of analogs of F-244 on HMG-CoA synthase was also investigated.

Inhibition of hepatic cholesterol biosynthesis by a 3-hydroxy-3-methylglutaryl coenzyme A synthase inhibitor, 1233A, in mice.

We have studied the in vivo inhibition of hepatic sterol biosynthesis by 1233A, a specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase. Administration of the compound orally to mice resulted in a dose-dependent inhibition of [14C]acetate incorporation into sterols in liver, but did not exert any significant effect on [14C]mevalonate incorporation. The results indicate that the in vivo inhibition of sterol synthesis occurs only at pre-mevalonate enzymatic steps in the sterol biosynthetic pathway, thus being compatible with the presumed site of inhibition, HMG-CoA synthase. Moreover, owing to irreversible inactivation of the enzyme by 1233A, it was possible to detect in vivo effect on the enzyme by assays of its activity in cell-free extracts from livers; the drug-treatment also resulted in a similarly dose-dependent inhibition of HMG-CoA synthase activity. In contrast, acetoacetyl-CoA thiolase and HMG-CoA reductase, the enzymes also responsible for mevalonate synthesis in the pathway, did not show any significant change in activity. These results clearly demonstrate that the inhibition of hepatic sterol synthesis caused by 1233A is indeed due to selective inhibition of HMG-CoA synthase in the tissues.

Synthesis of 1233A analogs and their inhibitory activity against hydroxymethylglutaryl coenzyme A synthase.

Simple and efficient syntheses of 1233A analogs were developed and the inhibitory activity of the analogs against hydroxymethylglutaryl coenzyme A (HMG-CoA) synthase was determined. Study of the structure-activity relationships revealed that not only the geometry in beta-lactone moiety but also the length of the carbon side chain is important for inhibitory activity against HMG-CoA synthase.

Method of search for microbial inhibitors of mevalonate biosynthesis using animal cells.

A new screening method for specific inhibitors of mevalonate biosynthesis was established using Vero cells, an animal cell line. The cultures selected were those which inhibited the growth of Vero cells in the EAGLE's minimum essential medium supplemented with 2% calf serum (2% CS-MEM) but lacked inhibitory activity against the growth of cells in 2% CS-MEM supplemented with 1 mM mevalonate. By this screening method, inhibitors of the two enzymes involved in mevalonate biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase, were selected from about 11,000 soil isolates. The beta-lactone 1233A, a fungal metabolite, was found to be the first naturally occurring compound which inhibits HMG-CoA synthase specifically and strongly. Monacolins K and J, inhibitors of HMG-CoA reductase, were also detected and identified.

Biosynthesis of antibiotic 1233A (F-244) and preparation of [14C]1233A.

The biosynthesis of antibiotic 1233A (F-244) was studied by feeding 13C-labeled precursors to the producing organism, Scopulariopsis sp. F-244. 13C NMR spectroscopy established that 1233A is derived from 4 methionines and 7 acetates. Seven acetates are condensed to form a hexaketide and 4 methyl residues from methionine are introduced into the main skeleton. The beta-lactone is derived from the alpha-carboxylic acid of the hexaketide. Since methionine was efficiently incorporated into 1233A, radiolabeled 1233A was prepared biosynthetically by feeding [14C]methionine to the producer. As a result, [14C]1233A was obtained with high specific radioactivity (27.2 muCi/mumols).