OGDH and Bcl-xL loss causes synthetic lethality in glioblastoma.

Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the α-ketoglutarate dehydrogenase (OGDH) gene, which encodes an enzyme that is part of the tricarboxylic acid (TCA) cycle, as essential for GBM growth. Moreover, by combining transcriptome and metabolite screening analyses, we discovered that loss of function of OGDH by the clinically validated drug compound CPI-613 was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3 mimetic, ABT263) in patient-derived xenografts as well neurosphere GBM cultures. CPI-613-mediated energy deprivation drove an integrated stress response with an upregulation of the BH3-only domain protein, Noxa, in an ATF4-dependent manner, as demonstrated by genetic loss-of-function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with disruption of the TCA cycle might be a treatment strategy for GBM.

Citrate Synthase and OGDH as Potential Biomarkers of Atherosclerosis under Chronic Stress.

BACKGROUND: Pathological changes of the adrenal gland and the possible underlying molecular mechanisms are currently unclear in the case of atherosclerosis (AS) combined with chronic stress (CS). METHODS: New Zealand white rabbits were used to construct a CS and AS animal model. Proteomics and bioinformatics were employed to identify hub proteins in the adrenal gland related to CS and AS. Hub proteins were detected using immunohistochemistry, immunofluorescence assays, and Western blotting. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression of genes. In addition, a neural network model was constructed. The quantitative relationships were inferred by cubic spline interpolation. Enzymatic activity of mitochondrial citrate synthase and OGDH was detected by the enzymatic assay kit. Function of citrate synthase and OGDH with knockdown experiments in the adrenal cell lines was performed. Furthermore, target genes-TF-miRNA regulatory network was constructed. Coimmunoprecipitation (IP) assay and molecular docking study were used to detect the interaction between citrate synthase and OGDH. RESULTS: Two most significant hub proteins (citrate synthase and OGDH) that were related to CS and AS were identified in the adrenal gland using numerous bioinformatic methods. The hub proteins were mainly enriched in mitochondrial proton transport ATP synthase complex, ATPase activation, and the AMPK signaling pathway. Compared with the control group, the adrenal glands were larger and more disordered, irregular, and necrotic in the AS+CS group. The expression of citrate synthase and OGDH was higher in the AS+CS group than in the control group, both at the protein and mRNA levels (P < 0.05). There were strong correlations among the cross-sectional areas of adrenal glands, citrate synthase, and OGDH (P < 0.05) via Spearman's rho analysis, receiver operating characteristic curves, a neural network model, and cubic spline interpolation. Enzymatic activity of citrate synthase and OGDH increased under the situation of atherosclerosis and chronic stress. Through the CCK8 assay, the adrenal cell viability was downregulated significantly after the knockdown experiment of citrate synthase and OGDH. Target genes-TF-miRNA regulatory network presented the close interrelations among the predicted microRNA, citrate synthase and OGDH. After Coimmunoprecipitation (IP) assay, the result manifested that the citrate synthase and OGDH were coexpressed in the adrenal gland. The molecular docking study showed that the docking score of optimal complex conformation between citrate synthase and OGDH was -6.15 kcal/mol. CONCLUSION: AS combined with CS plays a significant role on the hypothalamic-pituitary-adrenal (HPA) axis, promotes adrenomegaly, increases the release of glucocorticoid (GC), and might enhance ATP synthesis and energy metabolism in the body through citrate synthase and OGDH gene targets, providing a potential research direction for future related explorations into this mechanism.

Knock-Out of DHTKD1 Alters Mitochondrial Respiration and Function, and May Represent a Novel Pathway in Cardiometabolic Disease Risk.

Cardiometabolic disease affects the majority of individuals worldwide. The metabolite α-aminoadipic acid (2-AAA) was identified as a biomarker of Type 2 Diabetes (T2D). However, the mechanisms underlying this association remain unknown. DHTKD1, a central gene in the 2-AAA pathway, has been linked to 2-AAA levels and metabolic phenotypes. However, relatively little is known about its function. Here we report that DHTKD1 knock-out (KO) in HAP-1 cells leads to impaired mitochondrial structure and function. Despite impaired mitochondrial respiration and less ATP production, normal cell proliferation rate is maintained, potentially through a series of compensatory mechanisms, including increased mitochondrial content and Akt activation, p38, and ERK signaling. Common variants in DHTKD1 associate with Type 2 Diabetes and cardiometabolic traits in large genome-wide associations studies. These findings highlight the vital role of DHTKD1 in cellular metabolism and establish DHTKD1-mediated mitochondrial dysfunction as a potential novel pathway in cardiometabolic disease.

IDH1 mutation contributes to myeloid dysplasia in mice by disturbing heme biosynthesis and erythropoiesis.

Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts. In erythroid cells of these mice, D-2-hydroxyglutarate, an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase activity and diminishes succinyl-coenzyme A (CoA) production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells, while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species that induce the cell death of IDH1-mutant erythroid cells. Our results clearly show the essential role of IDH1 in normal erythropoiesis and describe how its mutation leads to myeloid disorders. These data thus have important implications for the devising of new treatments for IDH-mutant tumors.

Dynamic Control of 4-Hydroxyisoleucine Biosynthesis by Modified l-Isoleucine Biosensor in Recombinant Corynebacterium glutamicum.

4-Hydroxyisoleucine (4-HIL), a promising drug for treating diabetes, can be synthesized from the self-produced l-isoleucine (Ile) by expressing the Ile dioxygenase gene ido in Corynebacterium glutamicum. However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O2, makes such de novo biosynthesis difficult to be fulfilled effectively under static engineering conditions. In this study, dynamic control of 4-HIL biosynthesis by the Ile biosensor Lrp-PbrnFE was researched. The native PbrnFE promoter of natural Ile biosensor was still weak even under Ile induction. Through tetA dual genetic selection, several modified stronger PbrnFEN promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of ido expression by modified Ile biosensors increased the 4-HIL titer from 24.7 mM to 28.9-74.4 mM. The best strain ST04 produced even a little more 4-HIL than the static strain SN02 overexpressing ido by the strong PtacM promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different PbrnFEN-controlled odhI decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O2 supply in ST04 by different combinations of PbrnFEN-odhI and PbrnFEN-vgb improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by PbrnFE7. This titer was higher than those of all the static metabolic engineered C. glutamicum strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL de novo biosynthesis in C. glutamicum.

Inhibition and Crystal Structure of the Human DHTKD1-Thiamin Diphosphate Complex.

DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, which is an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here, we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of 10 disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies.

Complex Mitochondrial Dysfunction Induced by TPP+-Gentisic Acid and Mitochondrial Translation Inhibition by Doxycycline Evokes Synergistic Lethality in Breast Cancer Cells.

The mitochondrion has emerged as a promising therapeutic target for novel cancer treatments because of its essential role in tumorigenesis and resistance to chemotherapy. Previously, we described a natural compound, 10-((2,5-dihydroxybenzoyl)oxy)decyl) triphenylphosphonium bromide (GA-TPP+C10), with a hydroquinone scaffold that selectively targets the mitochondria of breast cancer (BC) cells by binding to the triphenylphosphonium group as a chemical chaperone; however, the mechanism of action remains unclear. In this work, we showed that GA-TPP+C10 causes time-dependent complex inhibition of the mitochondrial bioenergetics of BC cells, characterized by (1) an initial phase of mitochondrial uptake with an uncoupling effect of oxidative phosphorylation, as previously reported, (2) inhibition of Complex I-dependent respiration, and (3) a late phase of mitochondrial accumulation with inhibition of α-ketoglutarate dehydrogenase complex (αKGDHC) activity. These events led to cell cycle arrest in the G1 phase and cell death at 24 and 48 h of exposure, and the cells were rescued by the addition of the cell-penetrating metabolic intermediates l-aspartic acid ß-methyl ester (mAsp) and dimethyl α-ketoglutarate (dm-KG). In addition, this unexpected blocking of mitochondrial function triggered metabolic remodeling toward glycolysis, AMPK activation, increased expression of proliferator-activated receptor gamma coactivator 1-alpha (pgc1α) and electron transport chain (ETC) component-related genes encoded by mitochondrial DNA and downregulation of the uncoupling proteins ucp3 and ucp4, suggesting an AMPK-dependent prosurvival adaptive response in cancer cells. Consistent with this finding, we showed that inhibition of mitochondrial translation with doxycycline, a broad-spectrum antibiotic that inhibits the 28 S subunit of the mitochondrial ribosome, in the presence of GA-TPP+C10 significantly reduces the mt-CO1 and VDAC protein levels and the FCCP-stimulated maximal electron flux and promotes selective and synergistic cytotoxic effects on BC cells at 24 h of treatment. Based on our results, we propose that this combined strategy based on blockage of the adaptive response induced by mitochondrial bioenergetic inhibition may have therapeutic relevance in BC.

Selective Inhibitors of AlkB Family of Nucleic Acid Demethylases.

The α-ketoglutarate-dependent (AlkB) superfamily of FeII/2-oxoglutarate (2-OG)-dependent dioxygenases consists of a unique class of nucleic acid repair enzymes that reversibly remove alkyl substituents from nucleobases through oxidative dealkylation. Recent studies have verified the involvement of AlkB dioxygenases in a variety of human diseases. However, the development of small organic molecules that can function as enzyme inhibitors to block the action of oxidative dealkylation is still in its infancy. These purposeful chemical motifs, if capable of influencing the dealkylation activity, would have a potential clinical value by controlling genetic information expression. In this Perspective, we will summarize some of the most updated inhibitors of AlkB family demethylases and hope to provide a thought for the follow-up screening optimization.

Design and synthesis of biotinylated Hexylselen as a probe to identify KGA allosteric inhibitors by a convenient biomolecular interaction assay.

Hexylselen is a novel submicromolar dual KGA/GDH inhibitor, which demonstrates potent inhibition of cancer cells with minimal toxicity. To further investigation its mechanism of action, we designed and synthesized its biotinylated derivative 2 as a novel probe. From commercially available starting material, 2 was obtained in 6 steps with 13.4% overall yield. It is notable that this practical synthetic route give a template for the preparation of unsymmetrical di-benzo[d][1,2]selenazol-3(2H)-ones. Based on probe 2, we developed a novel biomolecular interaction assay for convenient and reliable test of KGA allosteric inhibitors and confirmed that hexylselen as an allosteric inhibitor of KGA sharing the same binding pocket with BPTES but not with Ebselen via competitive experiments.

Inactivation of TCA cycle enhances Staphylococcus aureus persister cell formation in stationary phase.

Persister cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of cells in stationary phase. By enrichment from a transposon mutant library in S. aureus we identified mutants that in this growth phase displayed enhanced persister cell formation. We found that inactivation of either sucA or sucB, encoding the subunits of the α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (TCA cycle), increased survival to lethal concentrations of ciprofloxacin by 10-100 fold as did inactivation of other TCA cycle genes or atpA encoding a subunit of the F1F0 ATPase. In S. aureus, TCA cycle activity and gene expression are de-repressed in stationary phase but single cells with low expression may be prone to form persisters. While ATP levels were not consistently affected in high persister mutants they commonly displayed reduced membrane potential, and persistence was enhanced by a protein motive force inhibitor. Our results show that persister cell formation in stationary phase does not correlate with ATP levels but is associated with low membrane potential.

Inhibition of α-ketoglutarate dehydrogenase activity affects adventitious root growth in poplar via changes in GABA shunt.

MAIN CONCLUSION: Blocking α-ketoglutarate dehydrogenase results in up-regulation of γ-aminobutyric acid (GABA) shunt activity, and inhibits the growth of poplar adventitious roots (ARs), indicating that AR growth is closely associated with GABA shunt. γ-Aminobutyric acid (GABA) shunt starts from α-ketoglutarate in the tricarboxylic acid cycle, which is thought to represent the cross road between carbon and nitrogen metabolism. Previous studies (Araújo et al. 2012b, Plant Cell 24: 2328-2351) have shown that blocking α-ketoglutarate dehydrogenase (α-KGDH) affects the GABA shunt activity, and inhibits growth. However, its effects on the growth of adventitious roots (ARs) are unclear. In this study, the growth of ARs in tissue-cultured 84K poplar (Populus alba × Populus glandulosa cv. '84K') was significantly inhibited when succinyl phosphate (SP), a specific inhibitor of α-KGDH, was supplied. The inhibition of ARs was associated with significant changes in the levels of soluble sugars, organic acids, and amino acids, and was coupled with the up-regulation of the GABA shunt activity at the transcriptional and translational levels. Exogenous GABA also inhibited AR growth following the increase of the endogenous GABA level. Transcriptomic analyses further showed that genes related to cell wall carbon metabolism and phytohormone (indoleacetic acid, ABA, and ethylene) signaling were affected by the changes of GABA shunt activity, resulting from the α-KGDH inhibition. Thus, our study indicates that the inhibition of poplar AR growth by blocking α-KGDH is closely associated with GABA shunt, which would benefit a better understanding of GABA's roles in plant development and stress response.

Parapyruvate, an Impurity in Pyruvate Supplements, Induces Senescence in Human Fibroblastic Hs68 Cells via Inhibition of the α-Ketoglutarate Dehydrogenase Complex.

Commercial dietary supplements of calcium pyruvate claim to be beneficial for losing weight, increasing muscle endurance, and regulating metabolism. Most industrial preparations have some impurities, including parapyruvate. Parapyruvate is an inhibitor of the α-ketoglutarate dehydrogenase complex (KGDHC). However, the effect and mechanism of parapyruvate on cell senescence and the content of parapyruvate in the dietary supplements of calcium pyruvate are unknown. In this study, we prepared pure parapyruvate with a purity of 99.8 ± 0.1% and investigated its ability to inhibit KGDHC activity and affect fibroblast senescence. Parapyruvate dose-dependently decreased KGDHC activity, with an IC50 of 4.13 mM and induced Hs68 cell senescence. Calcium ions, a KGDHC activator, antagonized the senescent effects of parapyruvate. The parapyruvate content was 1.4 ± 0.1% to 10.6 ± 0.2% in five brands of calcium pyruvate supplements. In this study, we showed that parapyruvate strongly induces Hs68 cell senescence by inhibiting KGDHC activity. Because of its KGDHC inhibition activity, the parapyruvate content should be an important issue for the food safety of calcium pyruvate supplements.

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury, preventing inactivation of the 2-oxoglutarate dehydrogenase complex.

BACKGROUND AND PURPOSE: Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine. EXPERIMENTAL APPROACH: Interventional controlled experimental animal study was used. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion traumatic brain injury. Thiamine was administered 1 h prior to trauma; cortex was extracted for analysis 4 h and 3 d after trauma. KEY RESULTS: Increased expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor receptor 1 (TNF-R1) by 4 h was accompanied by a decrease in mitochondrial respiration with glutamate but neither with pyruvate nor succinate. Assays of TCA cycle flux-limiting 2-oxoglutarate dehydrogenase complex (OGDHC) and functionally linked enzymes (glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, malate dehydrogenase and malic enzyme) indicated that only OGDHC activity was decreased. Application of the OGDHC coenzyme precursor thiamine rescued the activity of OGDHC and restored mitochondrial respiration. These effects were not mediated by changes in the expression of the OGDHC sub-units (E1k and E3), suggesting post-translational mechanism of thiamine effects. By the third day after TBI, thiamine treatment also decreased expression of TNF-R1. Specific markers of unfolded protein response did not change in response to thiamine. CONCLUSION AND IMPLICATIONS: Our data point to OGDHC as a major site of damage in mitochondria upon traumatic brain injury, which is associated with neuroinflammation and can be corrected by thiamine. Further studies are required to evaluate the pathological impact of these findings in clinical settings.

Effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor, OdhI, involved in glutamate production in Corynebacterium glutamicum.

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (ODH) complex is negatively regulated by the unphosphorylated form of OdhI protein, which is critical for L-glutamate overproduction. We examined the potential impact of protein acylation at lysine (K)-132 of OdhI in C. glutamicum ATCC13032. The K132E succinylation-mimic mutation reduced the ability of OdhI to bind OdhA, the catalytic subunit of the ODH complex, which reduced the inhibition of ODH activity. In vitro succinylation of OdhI protein also reduced the ability to inhibit ODH, and the K132R mutation blocked the effect. These results suggest that succinylation at K132 may attenuate the OdhI function. Consistent with these results, the C. glutamicum mutant strain with OdhI-K132E showed decreased L-glutamate production. Our results indicated that not only phosphorylation but also succinylation of OdhI protein may regulate L-glutamate production in C. glutamicum.

α-Ketoglutarate dehydrogenase complex moonlighting: ROS signalling added to the list: An Editorial highlight for 'Reductions in the mitochondrial enzyme α-ketoglutarate dehydrogenase complex in neurodegenerative disease - beneficial or detrimental?'

Read the highlighted article 'Reductions in the mitochondrial enzyme α-ketoglutarate dehydrogenase complex in neurodegenerative disease - beneficial or detrimental?' on page 823.

Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase.

Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.

Inhibition of mitochondrial 2-oxoglutarate dehydrogenase impairs viability of cancer cells in a cell-specific metabolism-dependent manner.

2-Oxoglutarate dehydrogenase (OGDH) of the tricarboxylic acid (TCA) cycle is often implied to be inactive in cancer, but this was not experimentally tested. We addressed the question through specific inhibition of OGDH by succinyl phosphonate (SP). SP action on different cancer cells was investigated using indicators of cellular viability and reactive oxygen species (ROS), metabolic profiling and transcriptomics. Relative sensitivity of various cancer cells to SP changed with increasing SP exposure and could differ in the ATP- and NAD(P)H-based assays. Glioblastoma responses to SP revealed metabolic sub-types increasing or decreasing cellular ATP/NAD(P)H ratio under OGDH inhibition. Cancer cell homeostasis was perturbed also when viability indicators were SP-resistant, e.g. in U87 and N2A cells. The transcriptomics database analysis showed that the SP-sensitive cells, such as A549 and T98G, exhibit the lowest expression of OGDH compared to other TCA cycle enzymes, associated with higher expression of affiliated pathways utilizing 2-oxoglutarate. Metabolic profiling confirmed the dependence of cellular SP reactivity on cell-specific expression of the pathways. Thus, oxidative decarboxylation of 2-oxoglutarate is significant for the interdependent homeostasis of NAD(P)H, ATP, ROS and key metabolites in various cancer cells. Assessment of cell-specific responses to OGDH inhibition is of diagnostic value for anticancer strategies.

Mild mitochondrial metabolic deficits by α-ketoglutarate dehydrogenase inhibition cause prominent changes in intracellular autophagic signaling: Potential role in the pathobiology of Alzheimer's disease.

Brain activities of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) are reduced in Alzheimer's disease and other age-related neurodegenerative disorders. The goal of the present study was to test the consequences of mild impairment of KGDHC on the structure, protein signaling and dynamics (mitophagy, fusion, fission, biogenesis) of the mitochondria. Inhibition of KGDHC reduced its in situ activity by 23-53% in human neuroblastoma SH-SY5Y cells, but neither altered the mitochondrial membrane potential nor the ATP levels at any tested time-points. The attenuated KGDHC activity increased translocation of dynamin-related protein-1 (Drp1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) from the cytosol to the mitochondria, and promoted mitochondrial cytochrome c release. Inhibition of KGDHC also increased the negative surface charges (anionic phospholipids as assessed by Annexin V binding) on the mitochondria. Morphological assessments of the mitochondria revealed increased fission and mitophagy. Taken together, our results suggest the existence of the regulation of the mitochondrial dynamism including fission and fusion by the mitochondrial KGDHC activity via the involvement of the cytosolic and mitochondrial protein signaling molecules. A better understanding of the link among mild impairment of metabolism, induction of mitophagy/autophagy and altered protein signaling will help to identify new mechanisms of neurodegeneration and reveal potential new therapeutic approaches.

Directed Regulation of Multienzyme Complexes of 2-Oxo Acid Dehydrogenases Using Phosphonate and Phosphinate Analogs of 2-Oxo Acids.

2-Oxo acid dehydrogenase complexes are important metabolic checkpoints functioning at the intercept of sugar and amino acid degradation. This review presents a short summary of architectural, catalytic, and regulatory principles of the complexes structure and function, based on recent advances in studies of well-characterized family members. Special attention is given to use of synthetic phosphonate and phosphinate analogs of 2-oxo acids as selective and efficient inhibitors of the cognate complexes in biological systems of bacterial, plant, and animal origin. We summarize our own results concerning the application of synthetic analogs of 2-oxo acids in situ and in vivo to reveal functional interactions between 2-oxo acid dehydrogenase complexes and other components of metabolic networks specific to different cells and tissues. Based on our study of glutamate excitotoxicity in cultured neurons, we show how a modulation of metabolism by specific inhibition of its key reaction may be employed to correct pathologies. This approach is further developed in our study on the action of the phosphonate analog of 2-oxoglutarate in animals. The study revealed that upregulation of 2-oxoglutarate dehydrogenase complex is involved in animal stress response and may provide increased resistance to damaging effects, underlying so-called preconditioning. The presented analysis of published data suggests synthetic inhibitors of metabolic checkpoints as promising tools to solve modern challenges of systems biology, metabolic engineering, and medicine.

Benzothiophene carboxylate derivatives as novel allosteric inhibitors of branched-chain α-ketoacid dehydrogenase kinase.

The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 µM). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1/2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.