Thiamine analogues featuring amino-oxetanes as potent and selective inhibitors of pyruvate dehydrogenase.

Pyruvate dehydrogenase complex (PDHc) is suppressed in some cancer types but overexpressed in others. To understand its contrasting oncogenic roles, there is a need for selective PDHc inhibitors. Its E1-subunit (PDH E1) is a thiamine pyrophosphate (TPP)-dependent enzyme and catalyses the first and rate-limiting step of the complex. In a recent study, we reported a series of ester-based thiamine analogues as selective TPP-competitive PDH E1 inhibitors with low nanomolar affinity. However, when the ester linker was replaced with an amide for stability reasons, the binding affinity was significantly reduced. In this study, we show that an amino-oxetane bioisostere of the amide improves the affinity and maintains stability towards esterase-catalysed hydrolysis.

Identification of PDHX as a metabolic target for esophageal squamous cell carcinoma.

The metabolism in tumors is reprogrammed to meet its energetic and substrate demands. However, this metabolic reprogramming creates metabolic vulnerabilities, providing new opportunities for cancer therapy. Metabolic vulnerability as a therapeutic target in esophageal squamous cell carcinoma (ESCC) has not been adequately clarified. Here, we identified pyruvate dehydrogenase (PDH) component X (PDHX) as a metabolically essential gene for the cell growth of ESCC. PDHX expression was required for the maintenance of PDH activity and the production of ATP, and its knockdown inhibited the proliferation of cancer stem cells (CSCs) and in vivo tumor growth. PDHX was concurrently upregulated with the CD44 gene, a marker of CSCs, by co-amplification at 11p13 in ESCC tumors and these genes coordinately functioned in cancer stemness. Furthermore, CPI-613, a PDH inhibitor, inhibited the proliferation of CSCs in vitro and the growth of ESCC xenograft tumors in vivo. Thus, our study provides new insights related to the development of novel therapeutic strategies for ESCC by targeting the PDH complex-associated metabolic vulnerability.

Pyruvate dehydrogenase kinases (PDKs): an overview toward clinical applications.

Pyruvate dehydrogenase kinase (PDK) can regulate the catalytic activity of pyruvate decarboxylation oxidation via the mitochondrial pyruvate dehydrogenase complex, and it further links glycolysis with the tricarboxylic acid cycle and ATP generation. This review seeks to elucidate the regulation of PDK activity in different species, mainly mammals, and the role of PDK inhibitors in preventing increased blood glucose, reducing injury caused by myocardial ischemia, and inducing apoptosis of tumor cells. Regulations of PDKs expression or activity represent a very promising approach for treatment of metabolic diseases including diabetes, heart failure, and cancer. The future research and development could be more focused on the biochemical understanding of the diseases, which would help understand the cellular energy metabolism and its regulation by pharmacological effectors of PDKs.

Differences in the efficiency of 3-deazathiamine and oxythiamine pyrophosphates as inhibitors of pyruvate dehydrogenase complex and growth of HeLa cells in vitro.

Oxythiamine (OT) and 3-deazathiamine (DAT) are the antimetabolites of thiamine. The aim of study was to compare the effects of OT and DAT pyrophosphates (-PP) on the kinetics of mammalian pyruvate dehydrogenase complex (PDHC) and the in vitro culture of HeLa cells. The kinetic study showed that 3-deazathiamine pyrophosphate (DATPP) was a much stronger competitive inhibitor (Ki = 0.0026 µM) of PDHC than OTPP (Ki = 0.025 µM). Both Ki values were much lower versus K m for thiamine pyrophosphate (0.06 µM). However, DATPP added to the culture medium for the HeLa cells culture did not hamper the rate of cell growth and showed not significant impact on the viability of the cells, whereas OTPP and OT showed a significant cytostatic effect. The differences between the thiamine antivitamins in their effect on cell growth in vitro may be due to differences in physicochemical properties and difficulty in DAT transport across the cell membrane.

HUANGKUISIWUFANG inhibits pyruvate dehydrogenase to improve glomerular injury in anti-Thy1 nephritis model.

ETHNOPHARMACOLOGICAL RELEVANCE: Huangkuisiwufang (HKSWF) is composed of Abelmoschus manihot (L.) Medik., Astragalus mongholicus, Polygonum cuspidatum, Curcuma longa L. Abelmoschus Manihot (L.) Medik. has been widely used for the treatment of chronic renal disease, oral ulcers and burn in China for centuries (Committee of the Pharmacopoeia of PR China, 2010). Abelmoschus manihot (L.) Medik., Polygonum cuspidatum, Curcuma longa L. have been mainly applied in folk medicine for their therapeutic effects on diabetes, cancer, heart disease and other diseases. AIM OF THE STUDY: We aimed to investigate the renoprotective function of HKSWF in anti-Thy nephritis model and clarify the relevant mechanisms. MATERIALS AND METHODS: One week after the model of glomerulonephritis created by injecting anti-thymocyte serum (ATS), rats were treated with Huangkui capsule, enalapril or HKSWF by gavage for a period of 8 weeks. The therapeutic effect was evaluated by detection of proteinuria, plasma creatine, blood urea nitrogen (BUN), podocyte injury, glomerular accumulation of extracellular matrix (ECM) and the markers of oxidative stress and renal fibrosis. RNA Sequencing (RNA-seq), KEGG and western blotting analysis were performed to indicate the signaling pathway involved in the therapeutic effect of HKSWF. RESULTS: Nephritic rats presented the increase of BUN, serum creatinine (Scr), proteinuria, podocyte damage, glomerular fibrosis, Ang II type 1 receptor (AT1R), and the reduction of creatinine clearance (Ccr). In contrast, application of HKSWF to nephritic rats decreased the levels of BUN and proteinuria, promoted mesangial cell recovery and improved oxidative stress level and podocyte injury. KEGG analysis revealed that pyruvate metabolism was the most significantly upregulated pathway in rats treated with HKSWF compared to disease control group. Increased pyruvate dehydrogenase and PAI-1 caused by nephritis was inhibited by HKSWF interposition. Furthermore, dichloroacetate sodium (DCA), an agonist of pyruvate dehydrogenase, could stimulate PAI-1 expression, which was suppressed by HKSWF. CONCLUSION: Chinese herbal preparation HKSWF has remarkable curative effects on glomerulonephritis animals. HKSWF attenuates pyruvate dehydrogenase to improve glomerular injury.

Dual Inhibition of Pyruvate Dehydrogenase Complex and Respiratory Chain Complex Induces Apoptosis by a Mitochondria-Targeted Fluorescent Organic Arsenical in†vitro and in†vivo.

Based on the potential therapeutic value in targeting mitochondria and the fluorophore tracing ability, a fluorescent mitochondria-targeted organic arsenical PDT-PAO-F16 was fabricated, which not only visualized the cellular distribution, but also exerted anti-cancer activity in vitro and in vivo via targeting pyruvate dehydrogenase complex (PDHC) and respiratory chain complexes in mitochondria. In details, PDT-PAO-F16 mainly accumulated into mitochondria within hours and suppressed the activity of PDHC resulting in the inhibition of ATP synthesis and thermogenesis disorder. Moreover, the suppression of respiratory chain complex I and IV accelerated the mitochondrial dysfunction leading to caspase family-dependent apoptosis. In vivo, the acute promyelocytic leukemia was greatly alleviated in the PDT-PAO-F16 treated group in APL mice model. Our results demonstrated the organic arsenical precursor with fluorescence imaging and target-anticancer efficacy is a promising anticancer drug.

Inhibition of pyruvate dehydrogenase complex activity by 3-bromopyruvate affects blood platelets responses in type 2 diabetes.

BACKGROUND: Hyperactivation of blood platelets is an essential factor in the pathomechanism of diabetes-evoked angiopathies. The aim of this work was to investigate whether blood platelets hyperactivation resulting from type 2 diabetic hyperglycaemia-increased pyruvate dehydrogenase complex activity and excessive acetyl-CoA accumulation may be brought to the normal range by the enzyme inhibitors. METHODS: Platelets were isolated from the blood of 9 type 2 diabetic patients and 10 healthy donors. Effects of 3-bromopyruvate and 3-nitropropionate on pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase activities, as well as levels of acetyl-CoA, ATP, thiobarbituric acid reactive species and aggregation were assessed in non-activated and thrombin-activated platelets. RESULTS: In type 2 diabetic patients fasting plasma glucose and fructosamine levels were 61 and 64% higher, respectively, than in the healthy group (p < 0.001). In non-activated diabetic platelets PDHC activity, PDHC-E2, acetyl-CoA and ATP levels were 66, 70, 68 and 60%, higher, respectively, than in platelets from healthy controls (p < 0.01). 3-bromopyruvate (0.1 mM) decreased pyruvate dehydrogenase activity in healthy and diabetic platelets by 42% and 59%, respectively. Similar inhibitory effects were observed for acetyl-CoA and ATP levels, aggregation and TBARS accumulation rates. Succinate dehydrogenase activity was inhibited by 3-nitropropionate (10 mM) to 38 and 41% of control values in healthy and diabetic platelets, respectively, but affected neither function nor acetyl-CoA metabolism in platelets of both groups. CONCLUSIONS: These data indicate that inhibition of pyruvate dehydrogenase excessive activity in diabetic platelets by 3-bromopyruvate may normalise their functional parameters through adjustment of acetyl-CoA/ATP levels to control values. Platelets from blood of diabetic patients display higher activities of pyruvate dehydrogenase complex (PDHC), higher levels of dihydrolipoate transacetylase (DLAT, E2 subunit of PDHC) as well as higher levels of acetyl-CoA yielding greater ATP/ADP accumulation than in platelets of normoglycemic subjects. Therefore, in diabetic platelets, thrombin caused higher release of ATP/ADP triggering excessive production of reactive oxygen species (ROS) and stronger aggregation compared to control platelets. In diabetic platelets, relative excess of DLAT in PDHC made them highly susceptible to 3-bromopyruvate (3BrP) inhibition. Resulting limitation of acetyl-CoA provision by 3-BrP normalised activity of diabetic platelets.

Loss of human arylamine N-acetyltransferase I regulates mitochondrial function by inhibition of the pyruvate dehydrogenase complex.

Human arylamine N-acetyltransferase 1 (NAT1) has been widely reported to affect cancer cell growth and survival and recent studies suggest it may alter cell metabolism. In this study, the effects of NAT1 deletion on mitochondrial function was examined in 2 human cell lines, breast carcinoma MDA-MB-231 and colon carcinoma HT-29 cells. Using a Seahorse XFe96 Flux Analyzer, NAT1 deletion was shown to decrease oxidative phosphorylation with a significant loss in respiratory reserve capacity in both cell lines. There also was a decrease in glycolysis without a change in glucose uptake. The changes in mitochondrial function was due to a decrease in pyruvate dehydrogenase activity, which could be reversed with the pyruvate dehydrogenase kinase inhibitor dichloroacetate. In the MDA-MB-231 and HT-29 cells, pyruvate dehydrogenase activity was attenuated either by an increase in phosphorylation or a decrease in total protein expression. These results may help explain some of the cellular events that have been reported recently in cell and animal models of NAT1 deficiency.

The HGF-MET axis coordinates liver cancer metabolism and autophagy for chemotherapeutic resistance.

Notwithstanding the numerous drugs available for liver cancer, emerging evidence suggests that chemotherapeutic resistance is a significant issue. HGF and its receptor MET play critical roles in liver carcinogenesis and metastasis, mainly dependent on the activity of receptor tyrosine kinase. However, for unknown reasons, all HGF-MET kinase activity-targeted drugs have failed or have been suspended in clinical trials thus far. Macroautophagy/autophagy is a protective 'self-eating' process for resisting metabolic stress by recycling obsolete components, whereas the impact of autophagy-mediated reprogrammed metabolism on therapeutic resistance is largely unclear, especially in liver cancer. In the present study, we first observed that HGF stimulus facilitated the Warburg effect and glutaminolysis to promote biogenesis in multiple liver cancer cells. We then identified the pyruvate dehydrogenase complex (PDHC) and GLS/GLS1 as crucial substrates of HGF-activated MET kinase; MET-mediated phosphorylation inhibits PDHC activity but activates GLS to promote cancer cell metabolism and biogenesis. We further found that the key residues of kinase activity in MET (Y1234/1235) also constitute a conserved LC3-interacting region motif (Y1234-Y1235-x-V1237). Therefore, on inhibiting HGF-mediated MET kinase activation, Y1234/1235-dephosphorylated MET induced autophagy to maintain biogenesis for cancer cell survival. Moreover, we verified that Y1234/1235-dephosphorylated MET correlated with autophagy in clinical liver cancer. Finally, a combination of MET inhibitor and autophagy suppressor significantly improved the therapeutic efficiency of liver cancer in vitro and in mice. Together, our findings reveal an HGF-MET axis-coordinated functional interaction between tyrosine kinase signaling and autophagy, and establish a MET-autophagy double-targeted strategy to overcome chemotherapeutic resistance in liver cancer. Abbreviations: ALDO: aldolase, fructose-bisphosphate; CQ: chloroquine; DLAT/PDCE2: dihydrolipoamide S-acetyltransferase; EMT: epithelial-mesenchymal transition; ENO: enolase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLS/GLS1: glutaminase; GLUL/GS: glutamine-ammonia ligase; GPI/PGI: glucose-6-phosphate isomerase; HCC: hepatocellular carcinoma; HGF: hepatocyte growth factor; HK: hexokinase; LDH: lactate dehydrogenase; LIHC: liver hepatocellular carcinoma; LIR: LC3-interacting region; PDH: pyruvate dehydrogenase; PDHA1: pyruvate dehydrogenase E1 alpha 1 subunit; PDHX: pyruvate dehydrogenase complex component X; PFK: phosphofructokinase; PK: pyruvate kinase; RTK: receptor tyrosine kinase; TCGA: The Cancer Genome Atlas.

Design, synthesis and biological evaluation of novel inhibitors against cyanobacterial pyruvate dehydrogenase multienzyme complex E1.

Cyanobacterial pyruvate dehydrogenase multienzyme complex E1 (PDHc E1) is a potential target enzyme for finding inhibitors to control harmful cyanobacterial blooms. In this study, a series of novel triazole thiamin diphosphate (ThDP) analogs were designed and synthesized by modifying the substituent group of triazole ring and optimizing triazole-benzene linker as potential cyanobacterial PDHc E1 (Cy-PDHc E1) inhibitors. Their inhibitory activities against Cy-PDHc E1 in vitro and algicide activities in vivo were further examined. Most of these compounds exhibited prominent inhibitory activities against Cy-PDHc E1 (IC50 1.48-4.48 µM) and good algicide activities against Synechocystis PCC6803 (EC50 0.84-2.44 µM) and Microcystis aeruginosa FACHB905 (EC50 0.74-1.77 µM). Especially, compound 8d showed not only the highest inhibitory activity against Cy-PDHc E1 (IC50 1.48 µM), but also the most powerful inhibitory selectivity between Cy-PDHc E1 (inhibitory rate 98.90%) and porcine PDHc E1 (inhibitory rate only 9.54%). Furthermore, the potential interaction between compound 8d and Cy-PDHc E1 was analyzed by a molecular docking method and site-directed mutagenesis and enzymatic analysis and fluorescence spectral analysis. These results indicated that compound 8d could be used as a hit compound for further optimization and might have potential to be developed as a new algicide.

Altering the sensitivity of Escherichia coli pyruvate dehydrogenase complex to NADH inhibition by structure-guided design.

A sufficient supply of reducing equivalents is essential for obtaining the maximum yield of target products in anaerobic fermentation. The pyruvate dehydrogenase (PDH) complex controls the critical step in pyruvate conversion to acetyl-CoA and NADH. However, in anaerobic Escherichia coli, PDH residing in the dihydrolipoamide dehydrogenase (LPD) component is normally inactive due to inhibition by NADH. In this study, the protein engineering of LPD by structural analysis was explored to eliminate this inhibition. A novel IAA350/351/358VVV triple mutant was successfully verified to be more effective than other LPD mutants reported till date. Notably, PDH activity with the triple mutant at an [NADH]/[NAD+] ratio of 0.15 was still higher than that of the wild-type without NADH addition. The altered enzyme of the PDH complex was also active in the presence of such high NADH levels. This is the first study concerning protein engineering of PDH by structure-guided design. The presence and functional activity of such an NADH-insensitive PDH complex provides a useful metabolic element for fermentation products and has potential for biotechnological application.

Stabilization of the hypoxia-inducible transcription Factor-1 alpha (HIF-1α) in thiamine deficiency is mediated by pyruvate accumulation.

Vitamin B1, or thiamine is a critical enzyme cofactor required for metabolic function and energy production. Thiamine deficiency (TD) is common in various diseases, and results in severe neurological complications due to diminished mitochondrial function, oxidative stress, excitotoxicity and inflammation. These pathological sequelae result in apoptotic cell death in both neurons and astrocytes in distinct regions, in particular the thalamus and mammillary bodies. Comparable histological injuries in patients with hypoxia/ischemia (H/I) have also been described, suggesting a congruency between the cellular responses to these stresses. Analogous to H/I, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α) even without changes in physiological oxygen levels. However, the mechanism of HIF-1α stabilization in TD is currently unknown. Using a pyruvate assay, we have demonstrated that TD induces pyruvate accumulation in mouse primary astrocytes which correlates to an increase in HIF-1α expression. Additionally, we utilized an enzymatic assay for pyruvate dehydrogenase to demonstrate a reduction in catalytic activity during TD due to lack of available thiamine pyrophosphate cofactor, resulting in the observed pyruvate accumulation. Finally, a pyruvate kinase inhibitor which limited pyruvate accumulation was utilized to demonstrate the role of pyruvate accumulation in HIF-1α stabilization during TD. These results reveal that stabilization of HIF-1α protein in TD centralizes on pyruvate accumulation in mouse primary astrocytes due to metabolic disruption of PDH.

Targeting cancer metabolism through synthetic lethality-based combinatorial treatment strategies.

PURPOSE OF REVIEW: Targeting cancer metabolism for therapy has received much attention over the last decade with various small molecule inhibitors entering clinical trials. The present review highlights the latest strategies to target glucose and glutamine metabolism for cancer therapy with a particular emphasis on novel combinatorial treatment approaches. RECENT FINDINGS: Inhibitors of glucose, lactate, and glutamine transport and the ensuing metabolism are in preclinical to clinical trial stages of investigation. Recent advances in our understanding of cell-intrinsic and cell-extrinsic factors that dictate dependence on these targets have informed the development of rational, synthetic lethality-based strategies to exploit these metabolic vulnerabilities. SUMMARY: Cancer cells exhibit a number of metabolic alterations with functional consequences beyond that of sustaining cellular energetics and biosynthesis. Elucidating context-specific metabolic dependencies and their connections to oncogenic signaling and epigenetic programs in tumor cells represents a promising approach to identify new metabolic drug targets for cancer therapy.

A novel inhibitor of pyruvate dehydrogenase kinase stimulates myocardial carbohydrate oxidation in diet-induced obesity.

The pyruvate dehydrogenase complex (PDC) is a key control point of energy metabolism and is subject to regulation by multiple mechanisms, including posttranslational phosphorylation by pyruvate dehydrogenase kinase (PDK). Pharmacological modulation of PDC activity could provide a new treatment for diabetic cardiomyopathy, as dysregulated substrate selection is concomitant with decreased heart function. Dichloroacetate (DCA), a classic PDK inhibitor, has been used to treat diabetic cardiomyopathy, but the lack of specificity and side effects of DCA indicate a more specific inhibitor of PDK is needed. This study was designed to determine the effects of a novel and highly selective PDK inhibitor, 2((2,4-dihydroxyphenyl)sulfonyl) isoindoline-4,6-diol (designated PS10), on pyruvate oxidation in diet-induced obese (DIO) mouse hearts compared with DCA-treated hearts. Four groups of mice were studied: lean control, DIO, DIO + DCA, and DIO + PS10. Both DCA and PS10 improved glucose tolerance in the intact animal. Pyruvate metabolism was studied in perfused hearts supplied with physiological mixtures of long chain fatty acids, lactate, and pyruvate. Analysis was performed using conventional 1H and 13C isotopomer methods in combination with hyperpolarized [1-13C]pyruvate in the same hearts. PS10 and DCA both stimulated flux through PDC as measured by the appearance of hyperpolarized [13C]bicarbonate. DCA but not PS10 increased hyperpolarized [1-13C]lactate production. Total carbohydrate oxidation was reduced in DIO mouse hearts but increased by DCA and PS10, the latter doing so without increasing lactate production. The present results suggest that PS10 is a more suitable PDK inhibitor for treatment of diabetic cardiomyopathy.

Targeting hepatic pyruvate dehydrogenase kinases restores insulin signaling and mitigates ChREBP-mediated lipogenesis in diet-induced obese mice.

OBJECTIVE: Mitochondrial pyruvate dehydrogenase kinases 1-4 (PDKs1-4) negatively regulate activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDKs play a pivotal role in maintaining energy homeostasis and contribute to metabolic flexibility by attenuating PDC activity in various mammalian tissues. Cumulative evidence has shown that the up-regulation of PDK4 expression is tightly associated with obesity and diabetes. In this investigation, we test the central hypothesis that PDKs1-4 are a pharmacological target for lowering glucose levels and restoring insulin sensitivity in obesity and type 2 diabetes (T2D). METHODS: Diet-induced obese (DIO) mice were treated with a liver-specific pan-PDK inhibitor 2-[(2,4-dihydroxyphenyl) sulfonyl]isoindoline-4,6-diol (PS10) for four weeks, and results compared with PDK2/PDK4 double knockout (DKO) mice on the same high fat diet (HFD). RESULTS: Both PS10-treated DIO mice and HFD-fed DKO mice showed significantly improved glucose, insulin and pyruvate tolerance, compared to DIO controls, with lower plasma insulin levels and increased insulin signaling in liver. In response to lower glucose levels, phosphorylated AMPK in PS10-treated DIO and HFD-fed DKO mice is upregulated, accompanied by decreased nuclear carbohydrate-responsive element binding protein (ChREBP). The reduced ChREBP signaling correlates with down-regulation of hepatic lipogenic enzymes (ACC1, FAS, and SCD1), leading to markedly diminished hepatic steatosis in both study groups, with lower circulating cholesterol and triacylglyceride levels as well as reduced fat mass. PS10-treated DIO as well as DKO mice showed predominant fatty acid over glucose oxidation. However, unlike systemic DKO mice, increased hepatic PDC activity alone in PS10-treated DIO mice does not raise the plasma total ketone body level. CONCLUSION: Our findings establish that specific targeting of hepatic PDKs with the PDK inhibitor PS10 is an effective therapeutic approach to maintaining glucose and lipid homeostasis in obesity and T2D, without the harmful ketoacidosis associated with systemic inhibition of PDKs.

Observation of acetyl phosphate formation in mammalian mitochondria using real-time in-organelle NMR metabolomics.

Recent studies point out the link between altered mitochondrial metabolism and cancer, and detailed understanding of mitochondrial metabolism requires real-time detection of its metabolites. Employing heteronuclear 2D NMR spectroscopy and 13C3-pyruvate, we propose in-organelle metabolomics that allows for the monitoring of mitochondrial metabolic changes in real time. The approach identified acetyl phosphate from human mitochondria, whose production has been largely neglected in eukaryotic metabolism since its first description about 70 years ago in bacteria. The kinetic profile of acetyl phosphate formation was biphasic, and its transient nature suggested its role as a metabolic intermediate. The method also allowed for the estimation of pyruvate dehydrogenase (PDH) enzyme activity through monitoring of the acetyl-CoA formation, independent of competing cytosolic metabolism. The results confirmed the positive regulation of mitochondrial PDH activity by p53, a well-known tumor suppressor. Our approach can easily be applied to other organelle-specific metabolic studies.

Acute activation of pyruvate dehydrogenase increases glucose oxidation in muscle without changing glucose uptake.

Pyruvate dehydrogenase (PDH) activity is a key component of the glucose/fatty acid cycle hypothesis for the regulation of glucose uptake and metabolism. We have investigated whether acute activation of PDH in muscle can alleviate the insulin resistance caused by feeding animals a high-fat diet (HFD). The importance of PDH activity in muscle glucose disposal under insulin-stimulated conditions was determined by infusing the PDH kinase inhibitor dichloroacetate (DCA) into HFD-fed Wistar rats during a hyperinsulinemic-euglycemic clamp. Acute DCA infusion did not alter glucose infusion rate, glucose disappearance, or hepatic glucose production but did decrease plasma lactate levels. DCA substantially increased muscle PDH activity; however, this did not improve insulin-stimulated glucose uptake in insulin-resistant muscle of HFD rats. DCA infusion increased the flux of pyruvate to acetyl-CoA and reduced glucose incorporation into glycogen and alanine in muscle. Similarly, in isolated muscle, DCA treatment increased glucose oxidation and decreased glycogen synthesis without changing glucose uptake. These results suggest that, although PDH activity controls the conversion of pyruvate to acetyl-CoA for oxidation, this has little effect on glucose uptake into muscle under insulin-stimulated conditions.

Actions of p-synephrine on hepatic enzyme activities linked to carbohydrate metabolism and ATP levels in vivo and in the perfused rat liver.

p-Synephrine is one of the main active components of the fruit of Citrus aurantium (bitter orange). Extracts of the bitter orange and other preparations containing p-synephrine have been used worldwide to promote weight loss and for sports performance. The purpose of the study was to measure the action of p-synephrine on hepatic enzyme activities linked to carbohydrate and energy metabolism and the levels of adenine mononucleotides. Enzymes and adenine mononucleotides were measured in the isolated perfused rat liver and in vivo after oral administration of the drug (50 and 300 mg/kg) by using standard techniques. p-Synephrine increased the activity of glycogen phosphorylase in vivo and in the perfused liver. It decreased, however, the activities of pyruvate kinase and pyruvate dehydrogenase also in vivo and in the perfused liver. p-Synephrine increased the hepatic pools of adenosine diphosphate and adenosine triphosphate. Stimulation of glycogen phosphorylase is consistent with the reported increased glycogenolysis in the perfused liver and increased glycemia in rats. The decrease in the pyruvate dehydrogenase activity indicates that p-synephrine is potentially capable of inhibiting the transformation of carbohydrates into lipids. The capability of increasing the adenosine triphosphate-adenosine diphosphate pool indicates a beneficial effect of p-synephrine on the cellular energetics.

Therapeutic Targeting of the Pyruvate Dehydrogenase Complex/Pyruvate Dehydrogenase Kinase (PDC/PDK) Axis in Cancer.

The mitochondrial pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl coenzyme A, thereby linking glycolysis to the tricarboxylic acid cycle and defining a critical step in cellular bioenergetics. Inhibition of PDC activity by pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation has been associated with the pathobiology of many disorders of metabolic integration, including cancer. Consequently, the PDC/PDK axis has long been a therapeutic target. The most common underlying mechanism accounting for PDC inhibition in these conditions is post-transcriptional upregulation of one or more PDK isoforms, leading to phosphorylation of the E1α subunit of PDC. Such perturbations of the PDC/PDK axis induce a "glycolytic shift," whereby affected cells favor adenosine triphosphate production by glycolysis over mitochondrial oxidative phosphorylation and cellular proliferation over cellular quiescence. Dichloroacetate is the prototypic xenobiotic inhibitor of PDK, thereby maintaining PDC in its unphosphorylated, catalytically active form. However, recent interest in the therapeutic targeting of the PDC/PDK axis for the treatment of cancer has yielded a new generation of small molecule PDK inhibitors. Ongoing investigations of the central role of PDC in cellular energy metabolism and its regulation by pharmacological effectors of PDKs promise to open multiple exciting vistas into the biochemical understanding and treatment of cancer and other diseases.

Design and optimization of N-acylhydrazone pyrimidine derivatives as E. coli PDHc E1 inhibitors: Structure-activity relationship analysis, biological evaluation and molecular docking study.

By targeting the thiamin diphosphate (ThDP) binding site of Escherichia coli (E. coli) pyruvate dehydrogenase multienzyme complex E1 (PDHc E1), a series of novel 'open-chain' classes of ThDP analogs A, B, and C with N-acylhydrazone moieties was designed and synthesized to explore their activities against E. coli PHDc E1 in vitro and their inhibitory activity against microbial diseases were further evaluated in vivo. As a result, A1-23 exhibited moderate to potent inhibitory activities against E. coli PDHc E1 (IC50=0.15-23.55µM). The potent inhibitors A13, A14, A15, C2, had strong inhibitory activities with IC50 values of 0.60, 0.15, 0.39 and 0.34µM against E. coli PDHc E1 and with good enzyme-selective inhibition between microorganisms and mammals. Especially, the most powerful inhibitor A14 could 99.37% control Xanthimonas oryzae pv. Oryzae. Furthermore, the binding features of compound A14 within E. coli PDHc E1 were investigated to provide useful insights for the further construction of new inhibitor by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that A14 had most powerful inhibition against E. coli PDHc E1 due to the establishment of stronger interaction with Glu571, Met194, Glu522, Leu264 and Phe602 at active site of E.coli PDHc E1. It could be used as a lead compound for further optimization, and may have potential as a new microbicide.