MERISTEM-DEFECTIVE regulates the balance between stemness and differentiation in the root meristem through RNA splicing control.

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of gene regulation is RNA alternative splicing. However, the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF) Arabidopsis gene encodes an SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast Snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and to repress stress response, cell differentiation and cell death pathways.

Lipid metabolism: a central modulator of RORt-mediated Th17 cell differentiation.

Among the T helper cell subsets, Th17 cells contribute to the development of various inflammatory and autoimmune diseases, including psoriasis, rheumatoid arthritis, inflammatory bowel disease, steroid-resistant asthma, and multiple sclerosis. Retinoid-related orphan receptor gamma t (RORγt), a nuclear hormone receptor, serves as a master transcription factor for Th17 cell differentiation. Recent findings have shown that modulating the metabolic pathway is critical for Th17 cell differentiation, particularly through the engagement of de novo lipid biosynthesis. Suppression of lipid biosynthesis, either through the pharmacological inhibition or gene deletion of related enzymes in CD4+ T cells, results in significant impairment of Th17 cell differentiation. Mechanistic studies indicate that metabolic fluxes through both the fatty acid and cholesterol biosynthetic pathways have a pivotal role in the regulation of RORγt activity through the generation of endogenous RORγt lipid ligands. This review discusses recent discoveries highlighting the importance of lipid metabolism in Th17 cell differentiation and function, as well as exploring specific molecular pathways involved in RORγt activation through cellular lipid metabolism. We further elaborate on a pioneering therapeutic approach to improve inflammatory and autoimmune disorders via the inhibition of RORγt.

Pin1 Exacerbates Non-Alcoholic Fatty Liver Disease by Enhancing Its Activity through Binding to ACC1.

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by diffuse hepatocellular steatosis due to fatty deposits in hepatocytes, excluding alcohol and other known liver injury factors. However, there are no specific drugs for the clinical treatment of NAFLD. Therefore, research on the pathogenesis of NAFLD at the cellular and molecular levels is a promising approach to finding therapeutic targets and developing targeted drugs for NAFLD. Pin1 is highly expressed during adipogenesis and contributes to adipose differentiation, but its specific mechanism of action in NAFLD is unclear. In this study, we investigated the role of Pin1 in promoting the development of NAFLD and its potential mechanisms in vitro and in vivo. First, Pin1 was verified in the NAFLD model in vitro using MCD diet-fed mice by Western Blot, RT-qPCR and immunohistochemistry (IHC) assays. In the in vitro study, we used the oleic acid (OA) stimulation-induced lipid accumulation model and examined the lipid accumulation in each group of cells by oil red O staining as well as BODIPY staining. The results showed that knockdown of Pin1 inhibited lipid accumulation in hepatocytes in an in vitro lipid accumulation model and improved lipid indices and liver injury levels. Moreover, in vivo, WT and Pin1-KO mice were fed a methionine-choline deficient (MCD) diet for 4 weeks to induce the NAFLD model. The effects of Pin1 on lipid accumulation, hepatic fibrosis, and oxidative stress were evaluated by biochemical analysis, glucose and insulin tolerance tests, histological analysis, IHC, RT-qPCR and Western blot assays. The results indicate that Pin1 knockdown significantly alleviated hepatic steatosis, fibrosis and inflammation in MCD-induced NAFLD mice, improved glucose tolerance and alleviated insulin resistance in mice. Further studies showed that the AMPK/ACC1 signalling pathway might take part in the process by which Pin1 regulates NAFLD, as evidenced by the inhibition of the AMPK/ACC1 pathway. In addition, immunofluorescence (IF), coimmunoprecipitation (Co-IP) and GST pull-down experiments also showed that Pin1 interacts directly with ACC1 and inhibits ACC1 phosphorylation levels. Our study suggests that Pin1 promotes NAFLD progression by inhibiting the activation of the AMPK/ACC1 signalling pathway, and it is possible that this effect is achieved by Pin1 interacting with ACC1 and inhibiting the phosphorylation of ACC1.

DDIT4 Downregulation by siRNA Approach Increases the Activity of Proteins Regulating Fatty Acid Metabolism upon Aspirin Treatment in Human Breast Cancer Cells.

Repositioning of aspirin for a more effective breast cancer (BC) treatment requires identification of predictive biomarkers. However, the molecular mechanism underlying the anticancer activity of aspirin remains fully undefined. Cancer cells enhance de novo fatty acid (FA) synthesis and FA oxidation to maintain a malignant phenotype, and the mechanistic target of rapamycin (mTORC1) is required for lipogenesis. We, therefore, aimed to test if the expression of mTORC1 suppressor DNA damage-inducible transcript (DDIT4) affects the activity of main enzymes in FA metabolism after aspirin treatment. MCF-7 and MDA-MB-468 human BC cell lines were transfected with siRNA to downregulate DDIT4. The expression of carnitine palmitoyltransferase 1 A (CPT1A) and serine 79-phosphorylated acetyl-CoA carboxylase 1 (ACC1) were analyzed by Western Blotting. Aspirin enhanced ACC1 phosphorylation by two-fold in MCF-7 cells and had no effect in MDA-MB-468 cells. Aspirin did not change the expression of CPT1A in either cell line. We have recently reported DDIT4 itself to be upregulated by aspirin. DDIT4 knockdown resulted in 1.5-fold decreased ACC1 phosphorylation (dephosphorylation activates the enzyme), 2-fold increased CPT1A expression in MCF-7 cells, and 2.8-fold reduced phosphorylation of ACC1 following aspirin exposure in MDA-MB-468 cells. Thus, DDIT4 downregulation raised the activity of main lipid metabolism enzymes upon aspirin exposure which is an undesired effect as FA synthesis and oxidation are linked to malignant phenotype. This finding may be clinically relevant as DDIT4 expression has been shown to vary in breast tumors. Our findings justify further, more extensive investigation of the role of DDIT4 in aspirin's effect on fatty acid metabolism in BC cells.

Fatty acid metabolism in T-cell function and differentiation.

Immunometabolism has recently emerged as a field of study examining the intersection between immunology and metabolism. Studies in this area have yielded new findings on the roles of a diverse range of metabolic pathways and metabolites, which have been found to control many aspects of T-cell biology, including cell differentiation, function and fate. A particularly important finding has been the discovery that to meet the energy requirements associated with their proliferation, activation and specific functions, T cells switch their metabolic signatures during differentiation. For example, whereas the induction of de novo fatty acid biosynthesis and fatty acid uptake programs are required for antigen-stimulation-induced proliferation and differentiation of effector T cells, fatty acid catabolism via ß-oxidation is essential for the generation of memory T cells and the differentiation of regulatory T cells. In this review, we discuss recent advances in our understanding of the metabolism in different stages of T cells and how fatty acid metabolism in these cells controls their specific functions.

[Knockdown of ACC1 promotes migration of esophageal cancer cell].

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 µmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.

MicroRNA-206 inhibits HCV proliferation through depressing ACC1 lipid synthesis signalling pathway.

MicroRNAs are considered to play important roles in cell biological and pathological progress. microRNA-206 (miR-206) was reported to participate in lipogenesis, and lipid droplets were necessary for the life cycle of HCV proliferation. Whether miR-206 was associated with HCV proliferation and the potential mechanism are not clear. In this study, we firstly identified that miR-206 could inhibit HCV proliferation at the RNA and protein level. Bioinformatical prediction of target genes binding to miR-206 was performed to investigate whether inhibiting function was due to a lipogenesis pathway. Then, the acetyl-CoA carboxylase 1 (ACC1) gene was selected as target gene of miR-206. The dual-luciferase reporter assay results showed that luciferase significantly decreased in cells transfected with 3'-UTR of the ACC1 gene and miR-206. The RNA and protein levels of the ACC1 gene and its pathway genes were significantly lower in cells transfected with miR-206 than in controls. Furthermore, the lipid droplet numbers also significantly decreased in cells transfected with miR-206. In conclusion, miR-206 could inhibit HCV proliferation through depressing ACC1 lipogenesis pathway and decreasing the lipid droplet numbers. miR-206 might be used as anti-HCV biochemical drug in the future.

On '1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis' by Yeong-Biau Yu, Douglas O.Adams and Shang Fa Yang.

The significance of the paper by Yu et al. (1979) is discussed in the context of the long history of ethylene as a plant growth regulator. By launching the era of molecular analysis and biotechnological exploitation, this research made a vital contribution to crop production and quality.

Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission.

Pluripotent stem cells are known to display distinct metabolic phenotypes than their somatic counterparts. While accumulating studies are focused on the roles of glucose and amino acid metabolism in facilitating pluripotency, little is known regarding the role of lipid metabolism in regulation of stem cell activities. Here, we show that fatty acid (FA) synthesis activation is critical for stem cell pluripotency. Our initial observations demonstrated enhanced lipogenesis in pluripotent cells and during cellular reprogramming. Further analysis indicated that de novo FA synthesis controls cellular reprogramming and embryonic stem cell pluripotency through mitochondrial fission. Mechanistically, we found that de novo FA synthesis regulated by the lipogenic enzyme ACC1 leads to the enhanced mitochondrial fission via (i) consumption of AcCoA which affects acetylation-mediated FIS1 ubiquitin-proteasome degradation and (ii) generation of lipid products that drive the mitochondrial dynamic equilibrium toward fission. Moreover, we demonstrated that the effect of Acc1 on cellular reprogramming via mitochondrial fission also exists in human iPSC induction. In summary, our study reveals a critical involvement of the FA synthesis pathway in promoting ESC pluripotency and iPSC formation via regulating mitochondrial fission.

Translational control of lipogenic enzymes in the cell cycle of synchronous, growing yeast cells.

Translational control during cell division determines when cells start a new cell cycle, how fast they complete it, the number of successive divisions, and how cells coordinate proliferation with available nutrients. The translational efficiencies of mRNAs in cells progressing synchronously through the mitotic cell cycle, while preserving the coupling of cell division with cell growth, remain uninvestigated. We now report comprehensive ribosome profiling of a yeast cell size series from the time of cell birth, to identify mRNAs under periodic translational control. The data reveal coordinate translational activation of mRNAs encoding lipogenic enzymes late in the cell cycle including Acc1p, the rate-limiting enzyme acetyl-CoA carboxylase. An upstream open reading frame (uORF) confers the translational control of ACC1 and adjusts Acc1p protein levels in different nutrients. The ACC1 uORF is relevant for cell division because its ablation delays cell cycle progression, reduces cell size, and suppresses the replicative longevity of cells lacking the Sch9p protein kinase regulator of ribosome biogenesis. These findings establish an unexpected relationship between lipogenesis and protein synthesis in mitotic cell divisions.

Design and synthesis of a monocyclic derivative as a selective ACC1 inhibitor by chemical modification of biphenyl ACC1/2 dual inhibitors.

A structure-activity relationship (SAR) study towards novel ACC1-selective inhibitors was carried out by modifying the molecular length of the linker in biaryl derivative 1 g, an ACC1/2 dual inhibitor. Ultimately, this leads us to discover novel phenoxybenzyloxy derivative 1i as a potent ACC1-selective inhibitor. Further chemical modification of this scaffold to improve cellular potency as well as physicochemical and pharmacokinetic (PK) properties produced N-2-(pyridin-2-ylethyl)acetamide derivative 1n, which showed highly potent ACC1-selective inhibition as well as sufficient PK profile for further in vivo evaluations. Oral administration of 1n significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of 100 mg/kg. Accordingly, our novel series of potent ACC1-selective inhibitors represents a set of useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid-related diseases.

Overexpression and repression of key rate-limiting enzymes (acetyl CoA carboxylase and HMG reductase) to enhance fatty acid production from Rhodotorula mucilaginosa.

Implementing two-way strategies to enhance the lipid production in Rhodotorula mucilaginosa with the help of metabolic engineering was focused on the overexpression of acetyl coenzyme A carboxylase (ACC1 carboxylase) gene and repression of 3-hydroxy 3-methylglutaryl reductase (HMG-CoA reductase). Using an inducer (sodium citrate) and inhibitor (rosuvastatin), the amounts of biomass, lipid, and carotenoid were estimated. In the presence of inhibitor (200 mM), 62% higher lipid concentration was observed, while 44% enhancement was recorded when inducer (3 mM) was used. A combination of both inhibitor and inducer resulted in a 57% increase in lipid concentration by the oleaginous yeast. These results were again confirmed by real-time polymerase chain reaction by targeting the expression of the genes coding for ACC1 carboxylase and 13-fold increase was recorded in the presence of inducer as compared with control. This combined strategy (inducer and inhibitor use) has been reported for the first time as far as the best of our knowledge. The metabolic engineering strategies reported here will be a powerful approach for the enhanced commercial production of lipids.

Acetyl-CoA carboxylase 1-dependent lipogenesis promotes autophagy downstream of AMPK.

Autophagy, a membrane-dependent catabolic process, ensures survival of aging cells and depends on the cellular energetic status. Acetyl-CoA carboxylase 1 (Acc1) connects central energy metabolism to lipid biosynthesis and is rate-limiting for the de novo synthesis of lipids. However, it is unclear how de novo lipogenesis and its metabolic consequences affect autophagic activity. Here, we show that in aging yeast, autophagy levels highly depend on the activity of Acc1. Constitutively active Acc1 (acc1S/A ) or a deletion of the Acc1 negative regulator, Snf1 (yeast AMPK), shows elevated autophagy levels, which can be reversed by the Acc1 inhibitor soraphen A. Vice versa, pharmacological inhibition of Acc1 drastically reduces cell survival and results in the accumulation of Atg8-positive structures at the vacuolar membrane, suggesting late defects in the autophagic cascade. As expected, acc1S/A cells exhibit a reduction in acetate/acetyl-CoA availability along with elevated cellular lipid content. However, concomitant administration of acetate fails to fully revert the increase in autophagy exerted by acc1S/A Instead, administration of oleate, while mimicking constitutively active Acc1 in WT cells, alleviates the vacuolar fusion defects induced by Acc1 inhibition. Our results argue for a largely lipid-dependent process of autophagy regulation downstream of Acc1. We present a versatile genetic model to investigate the complex relationship between acetate metabolism, lipid homeostasis, and autophagy and propose Acc1-dependent lipogenesis as a fundamental metabolic path downstream of Snf1 to maintain autophagy and survival during cellular aging.

SIRT3 promotes the invasion and metastasis of cervical cancer cells by regulating fatty acid synthase.

Sirtuin 3 (SIRT3) modulates mitochondria-localized processes and is implicated in the metabolic reprogramming of cancer cells, especially fatty acid (FA) synthesis. However, the relationship between SIRT3 and aberrant lipid synthesis in cervical cancer remains unclear. Here, we investigated the clinical relevance of SIRT3 expression in cervical squamous cell carcinoma (CSCC), cervical intraepithelial neoplasia (CIN), and normal tissues. To analyze the role of SIRT3 in CCSC in vitro, endogenous SIRT3 levels were up- and down-regulated in SiHa and C33a cells, respectively, via lentiviral-based transfection. Levels were quantified using qRT-PCR. Acetylation levels for acetyl-coA carboxylase (ACC1) were measured with the anti-acetyllysine antibody. Knockdown of SIRT3 reduced levels of cellular lipid content in cells. To investigate the role of SIRT3 in cell proliferation, nude mice were xenografted with SIRT3-overexpressing or SIRT3-knockdown CCSC cells. Overall, the results demonstrate that SIRT3 significantly contributed to the reprogramming of FA synthesis in CCSC by up-regulating ACC1 to promote de novo lipogenesis by SIRT3 deacetylation. Moreover, the findings show that the SIRT3-mediated regulation of FA synthesis played a critical role in the proliferation and metastasis of CCSC cells, suggesting that SIRT3 has therapeutic potential in CCSC treatment.

Microglial activation in an amyotrophic lateral sclerosis-like model caused by Ranbp2 loss and nucleocytoplasmic transport impairment in retinal ganglion neurons.

Nucleocytoplasmic transport is dysregulated in sporadic and familial amyotrophic lateral sclerosis (ALS) and retinal ganglion neurons (RGNs) are purportedly involved in ALS. The Ran-binding protein 2 (Ranbp2) controls rate-limiting steps of nucleocytoplasmic transport. Mice with Ranbp2 loss in Thy1+-motoneurons develop cardinal ALS-like motor traits, but the impairments in RGNs and the degree of dysfunctional consonance between RGNs and motoneurons caused by Ranbp2 loss are unknown. This will help to understand the role of nucleocytoplasmic transport in the differential vulnerability of neuronal cell types to ALS and to uncover non-motor endophenotypes with pathognomonic signs of ALS. Here, we ascertain Ranbp2's function and endophenotypes in RGNs of an ALS-like mouse model lacking Ranbp2 in motoneurons and RGNs. Thy1+-RGNs lacking Ranbp2 shared with motoneurons the dysregulation of nucleocytoplasmic transport. RGN abnormalities were comprised morphologically by soma hypertrophy and optic nerve axonopathy and physiologically by a delay of the visual pathway's evoked potentials. Whole-transcriptome analysis showed restricted transcriptional changes in optic nerves that were distinct from those found in sciatic nerves. Specifically, the level and nucleocytoplasmic partition of the anti-apoptotic and novel substrate of Ranbp2, Pttg1/securin, were dysregulated. Further, acetyl-CoA carboxylase 1, which modulates de novo synthesis of fatty acids and T-cell immunity, showed the highest up-regulation (35-fold). This effect was reflected by the activation of ramified CD11b+ and CD45+-microglia, increase of F4\80+-microglia and a shift from pseudopodial/lamellipodial to amoeboidal F4\80+-microglia intermingled between RGNs of naive mice. Further, there was the intracellular sequestration in RGNs of metalloproteinase-28, which regulates macrophage recruitment and polarization in inflammation. Hence, Ranbp2 genetic insults in RGNs and motoneurons trigger distinct paracrine signaling likely by the dysregulation of nucleocytoplasmic transport of neuronal-type selective substrates. Immune-modulators underpinning RGN-to-microglial signaling are regulated by Ranbp2, and this neuronal-glial system manifests endophenotypes that are likely useful in the prognosis and diagnosis of motoneuron diseases, such as ALS.

Disruption of beta cell acetyl-CoA carboxylase-1 in mice impairs insulin secretion and beta cell mass.

AIMS/HYPOTHESIS: Pancreatic beta cells secrete insulin to maintain glucose homeostasis, and beta cell failure is a hallmark of type 2 diabetes. Glucose triggers insulin secretion in beta cells via oxidative mitochondrial pathways. However, it also feeds mitochondrial anaplerotic pathways, driving citrate export and cytosolic malonyl-CoA production by the acetyl-CoA carboxylase 1 (ACC1) enzyme. This pathway has been proposed as an alternative glucose-sensing mechanism, supported mainly by in vitro data. Here, we sought to address the role of the beta cell ACC1-coupled pathway in insulin secretion and glucose homeostasis in vivo. METHODS: Acaca, encoding ACC1 (the principal ACC isoform in islets), was deleted in beta cells of mice using the Cre/loxP system. Acaca floxed mice were crossed with Ins2cre mice (ßACC1KO; life-long beta cell gene deletion) or Pdx1creER mice (tmx-ßACC1KO; inducible gene deletion in adult beta cells). Beta cell function was assessed using in vivo metabolic physiology and ex vivo islet experiments. Beta cell mass was analysed using histological techniques. RESULTS: ßACC1KO and tmx-ßACC1KO mice were glucose intolerant and had defective insulin secretion in vivo. Isolated islet studies identified impaired insulin secretion from beta cells, independent of changes in the abundance of neutral lipids previously implicated as amplification signals. Pancreatic morphometry unexpectedly revealed reduced beta cell size in ßACC1KO mice but not in tmx-ßACC1KO mice, with decreased levels of proteins involved in the mechanistic target of rapamycin kinase (mTOR)-dependent protein translation pathway underpinning this effect. CONCLUSIONS/INTERPRETATION: Our study demonstrates that the beta cell ACC1-coupled pathway is critical for insulin secretion in vivo and ex vivo and that it is indispensable for glucose homeostasis. We further reveal a role for ACC1 in controlling beta cell growth prior to adulthood.

Structural Insights into Catalytic Relevances of Substrate Poses in ACC-1.

ACC-1 is a plasmid-encoded class C ß-lactamase identified in clinical isolates of Klebsiella pneumoniae, Proteus mirabilis, Salmonella enterica, and Escherichia coli ACC-1-producing bacteria are susceptible to cefoxitin, whereas they are resistant to oxyimino cephalosporins. Here, we depict crystal structures of apo ACC-1, adenylylated ACC-1, and acylated ACC-1 complexed with cefotaxime and cefoxitin. ACC-1 has noteworthy structural alterations in the R2 loop, the Ω loop, and the Phe119 loop located along the active-site rim. The adenylate covalently bonded to the nucleophilic serine reveals a tetrahedral phosphorus mimicking the deacylation transition state. Cefotaxime in ACC-1 has a proper conformation for the substrate-assisted catalysis in that its C-4 carboxylate and N-5 nitrogen are adequately located to facilitate the deacylation reaction. In contrast, cefoxitin in ACC-1 has a distinct conformation, in which those functional groups cannot contribute to catalysis. Furthermore, the orientation of the deacylating water relative to the acyl carbonyl group in ACC-1 is unfavorable for nucleophilic attack.

Genome constitution and evolution of Elytrigia lolioides inferred from Acc1, EF-G, ITS, TrnL-F sequences and GISH.

BACKGROUND: Elytrigia lolioides (Kar. et Kir.) Nevski, which is a perennial, cross-pollinating wheatgrass that is distributed in Russia and Kazakhstan, is classified into Elytrigia, Elymus, and Lophopyrum genera by taxonomists on the basis of different taxonomic classification systems. However, the genomic constitution of E. lolioides is still unknown. To identify the genome constitution and evolution of E. lolioides, we used single-copy nuclear genes acetyl-CoA carboxylase (Acc1) and elongation factor G (EF-G), multi-copy nuclear gene internal transcribed space (ITS), chloroplast gene trnL-F together with fluorescence and genomic in situ hybridization. RESULTS: Despite the widespread homogenization of ITS sequences, two distinct lineages (genera Pseudoroegneria and Hordeum) were identified. Acc1 and EF-G sequences suggested that in addition to Pseudoroegneria and Hordeum, unknown genome was the third potential donor of E. lolioides. Data from chloroplast DNA showed that Pseudoroegneria is the maternal donor of E. lolioides. Data from specific FISH marker for St genome indicated that E. lolioides has two sets of St genomes. Both genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) results confirmed the presence of Hordeum genome in this species. When E genome was used as the probe, no signal was found in 42 chromosomes. The E-like copy of Acc1 sequences was detected in E. lolioides possibly due to the introgression from E genome species. One of the H chromosomes in the accession W6-26586 from Kazakhstan did not hybridize H genome signals but had St genome signals on the pericentromeric regions in the two-color GISH. CONCLUSIONS: Phylogenetic and in situ hybridization indicated the presence of two sets of Pseudoroegneria and one set of Hordeum genome in E. lolioides. The genome formula of E. lolioides was designed as StStStStHH. E. lolioides may have originated through the hybridization between tetraploid Elymus (StH) and diploid Pseudoroegneria species. E and unknown genomes may participate in the speciation of E. lolioides through introgression. According to the genome classification system, E. lolioides should be transferred into Elymus L. and renamed as Elymus lolioidus (Kar. er Kir.) Meld.

The identification and pharmacological evaluation of potent, selective and orally available ACC1 inhibitor.

In our effort to explore the potential of ACC1-selective inhibitor as in vivo probe molecule, a series of 1,3-benzoxazole derivatives was synthesized. Previously, we reported a series of novel bicyclic and monocyclic ACC1-selective inhibitors. Among them, compound 1a exhibited highly potent cellular activity (acetate uptake IC50 = 0.76 nM) as well as promising in vivo PD efficacy. However, compound 1a caused severe body weight reduction in repeated dose administration in the mouse model. Since 1a showed potent inhibitory activity against mouse ACC1 as well as strong inhibition of mouse ACC2, we further examined a series of 1a analogues in order to reduce undesirable body weight change. The replacement of acetamide moiety with ureido moiety dramatically improved selectivity of mouse ACC1 against ACC2. In addition, analogue 1b displayed favorable bioavailability in mouse cassette dosing PK study, hence in vivo PD studies were also carried out. Oral administration of 1b significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of more than 30 mg/kg. Furthermore, compound 1b showed significant antitumor efficacy in 786-O xenograft mice at an oral dose of 30 mg/kg (T/C = 0.5%). Accordingly, our novel potent ACC1-selective inhibitor represents a set of useful orally-available research tools, as well as potential therapeutic agents particularly in terms of new cancer therapies.

Design and synthesis of a novel 1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative as an orally available ACC1 inhibitor.

We initiated our structure-activity relationship (SAR) studies for novel ACC1 inhibitors from 1a as a lead compound. Our initial SAR studies of 1H-Pyrrolo[3,2-b]pyridine-3-carboxamide scaffold revealed the participation of HBD and HBA for ACC1 inhibitory potency and identified 1-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1c as a potent ACC1 inhibitor. Although compound 1c had physicochemical and pharmacokinetic (PK) issues, we investigated the 1H-pyrrolo[3,2-b]pyridine core scaffold to address these issues. Accordingly, this led us to discover a novel 1-isopropyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1k as a promising ACC1 inhibitor, which showed potent ACC1 inhibition as well as sufficient cellular potency. Since compound 1k displayed favorable bioavailability in mouse cassette dosing PK study, we conducted in vivo Pharmacodynamics (PD) studies of this compound. Oral administration of 1k significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at a dose of 100 mg/kg. Accordingly, our novel series of potent ACC1 inhibitors represent useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid related diseases.