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Bioorganic & Medicinal Chemistry Letters Jun 2021Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the...
Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the metabolic processes in cancer cells and thus selectively delay or inhibit growth in transformed versus normal cells. We have previously disclosed a pyrazole-based series of potent LDH inhibitors with long residence times on the enzyme. Here, we report the elaboration of a new subseries of LDH inhibitors based on those leads. These new compounds potently inhibit both LDHA and LDHB enzymes, and inhibit lactate production in cancer cell lines.
Topics: Aniline Compounds; Antineoplastic Agents; Cell Line, Tumor; Drug Design; Ethers; Humans; L-Lactate Dehydrogenase
PubMed: 33771585
DOI: 10.1016/j.bmcl.2021.127974 -
Environmental Microbiology Aug 2021Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to...
Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoacetica. Growth and metabolism were dependent on CO and the chemiosmotic gradient. We discovered a l-lactate:NAD oxidoreductase (LDH) in cell-free extracts, exhibiting an average specific activity of 362.8 ± 22.9 mU mg . The enzyme was reversible, most active at 65°C and pH 9, with Km values of 23.1 ± 3.7 mM for l-lactate and 273.3 ± 39.1 μM for NAD . In-gel activity assays and mass spectrometric proteomics revealed that the ldh gene encoded the characterized LDH. Transcriptomic and genomic analyses showed that ldh expression was induced by lactate and there was a single nucleotide polymorphism near the predicted NAD binding site. Genes encoding central redox and energy metabolism complexes, such as, the energetic coupling site Ech2, menaquinone, and the electron bifurcating EtfABCX and MTHFR were also upregulated in cells grown on lactate. These findings ultimately lead to a redox-balanced metabolic model that shows how growth on lactate can proceed in a microorganism that only has a conventional NAD -reducing LDH.
Topics: Anaerobiosis; L-Lactate Dehydrogenase; Lactic Acid; NAD; Oxidation-Reduction
PubMed: 34190373
DOI: 10.1111/1462-2920.15657 -
Applied and Environmental Microbiology Jan 2021Growth of PCA on lactate was enhanced by laboratory adaptive evolution. The enhanced growth was considered to be attributed to increased expression of the genes,...
Growth of PCA on lactate was enhanced by laboratory adaptive evolution. The enhanced growth was considered to be attributed to increased expression of the genes, encoding a succinyl-coenzyme A (CoA) synthetase. To further investigate the function of the succinyl-CoA synthetase, the genes were deleted from The mutant showed defective growth on lactate but not on acetate. Introduction of the genes into the mutant restored the full potential to grow on lactate. These results verify the importance of the succinyl-CoA synthetase in growth on lactate. Genome analysis of species identified candidate genes, GSU1623, GSU1624, and GSU1620, for lactate dehydrogenase. Deletion mutants of the identified genes for d-lactate dehydrogenase (ΔGSU1623 ΔGSU1624 mutant) or l-lactate dehydrogenase (ΔGSU1620 mutant) could not grow on d-lactate or l-lactate but could grow on acetate and l- or d-lactate, respectively. Introduction of the respective genes into the mutants allowed growth on the corresponding lactate stereoisomer. These results suggest that the identified genes were essential for d- or l-lactate utilization. The reporter assay demonstrated that the putative promoter regions were more active during growth on lactate than during growth on acetate, indicating that the genes for the lactate dehydrogenases were expressed more during growth on lactate than during growth on acetate. The gene deletion phenotypes and the expression profiles indicate that there are metabolic switches between lactate and acetate. This study advances the understanding of anaerobic lactate utilization in Lactate is a microbial fermentation product as well as a source of carbon and electrons for microorganisms in the environment. Furthermore, lactate is a common amendment for stimulation of microbial growth in environmental biotechnology applications. However, anaerobic metabolism of lactate has been poorly studied for environmentally relevant microorganisms. species are found in various environments and environmental biotechnology applications. By employing genomic and genetic approaches, succinyl-CoA synthetase and lactate dehydrogenase were identified as key enzymes in anaerobic metabolism of lactate in , a representative species. Differential gene expression during growth on lactate and acetate was observed, demonstrating that could metabolically switch to adapt to available substrates in the environment. The findings provide new insights into basic physiology in lactate metabolism as well as cellular responses to growth conditions in the environment and can be informative for the application of lactate in environmental biotechnology.
Topics: Anaerobiosis; Bacterial Proteins; Gene Expression Regulation, Bacterial; Geobacter; L-Lactate Dehydrogenase; Lactic Acid; Succinate-CoA Ligases
PubMed: 33158892
DOI: 10.1128/AEM.01968-20 -
Development (Cambridge, England) Sep 2019The dramatic growth that occurs during larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands...
The dramatic growth that occurs during larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands by increasing carbohydrate metabolism and lactate dehydrogenase (LDH) activity. The resulting metabolic program is ideally suited for synthesis of macromolecules and mimics the manner by which cancer cells rely on aerobic glycolysis. To explore the potential role of LDH in promoting biosynthesis, we examined how mutations influence larval development. Our studies unexpectedly found that mutants grow at a normal rate, indicating that LDH is dispensable for larval biomass production. However, subsequent metabolomic analyses suggested that mutants compensate for the inability to produce lactate by generating excess glycerol-3-phosphate (G3P), the production of which also influences larval redox balance. Consistent with this possibility, larvae lacking both LDH and G3P dehydrogenase (GPDH1) exhibit growth defects, synthetic lethality and decreased glycolytic flux. Considering that human cells also generate G3P upon inhibition of lactate dehydrogenase A (LDHA), our findings hint at a conserved mechanism in which the coordinate regulation of lactate and G3P synthesis imparts metabolic robustness to growing animal tissues.
Topics: Adenosine Triphosphate; Animals; Animals, Genetically Modified; Drosophila melanogaster; Female; Glycerolphosphate Dehydrogenase; Glycolysis; Homeostasis; L-Lactate Dehydrogenase; Lactic Acid; Larva; Male; Mutation; NAD; Oxidation-Reduction; Sugars
PubMed: 31399469
DOI: 10.1242/dev.175315 -
Laboratory Medicine Sep 2022To evaluate the difference in lactate dehydrogenase (LDH) concentrations in plasma vs serum specimens in our patient population.
OBJECTIVE
To evaluate the difference in lactate dehydrogenase (LDH) concentrations in plasma vs serum specimens in our patient population.
MATERIALS AND METHODS
We measured LDH in 110 paired plasma and serum specimens over a 2-week period. Hemolytic indices were performed on each specimen. These paired specimens were drawn in a single setting and stored under the same conditions. For the last 14 paired specimens, cell counts were performed on the plasma/serum.
RESULTS
Plasma LDH was on average 22% higher than serum LDH. There was no difference in the hemolytic indices between the plasma and the serum specimens. In the last 14 specimens, cell counts revealed increased platelets in the plasma specimens compared to the serum specimens.
CONCLUSION
We propose switching back to using serum for LDH testing because there was unpredictable elevation in plasma LDH concentrations. These elevations in LDH levels may be linked to the platelets present in plasma and that may lyse or become activated with storage at refrigerated temperature.
Topics: Blood Platelets; Humans; L-Lactate Dehydrogenase; Plasma
PubMed: 35512404
DOI: 10.1093/labmed/lmac026 -
The Journal of Veterinary Medical... Oct 2022Lactate dehydrogenase (LDH) in blood is measured using the Japanese Society of Clinical Chemistry (JSCC) method in Japan and the International Federation of Clinical...
Lactate dehydrogenase (LDH) in blood is measured using the Japanese Society of Clinical Chemistry (JSCC) method in Japan and the International Federation of Clinical Chemistry (IFCC) method in other countries. In human clinical practice, the IFCC method replaced the JSCC method due to international standardization. Moreover, veterinary LDH measurement will also eventually shift to the IFCC method. However, the relationship between the IFCC and JSCC methods for LDH in various animals and whether they can be equated or not have not yet been investigated. This study aimed to present the changes in measurements in canines and felines after switching to the IFCC method. The plasma LDH activity of canines (N=177) and felines (N=115), who visited a secondary care veterinary clinic, was measured using the JSCC and IFCC methods. The IFCC/JSCC ratio was <1.0 in 85% of canines and 88% of felines, indicating that the IFCC method tended to show lower values than the JSCC method, presumably because LDH5 is dominant among the LDH isozymes in canines and felines. The increase in the systematic errors of both assays was in the high value range, with some samples exceeding the error tolerance from near the upper end of the reference range. When switching to the IFCC method for LDH measurement in canines and felines, each institution should consider whether the reference range and clinical diagnostic values established by the JSCC method are appropriate for continued use.
Topics: Animals; Cat Diseases; Cats; Dog Diseases; Dogs; Humans; Isoenzymes; L-Lactate Dehydrogenase; Reference Standards
PubMed: 35934797
DOI: 10.1292/jvms.22-0278 -
Applied Microbiology and Biotechnology Dec 2023Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of...
Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaC, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaC was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent L-valine depletion caused by dominant L-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent D-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent L-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaC, ldhA-hadA encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaC. The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaC functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell. KEY POINTS: (R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha (R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identified The engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB.
Topics: Cupriavidus necator; L-Lactate Dehydrogenase; Lactate Dehydrogenases; Cadmium; Hydroxybutyrates; Polyhydroxyalkanoates; Polyesters; Escherichia coli; Valine; Lactates; Glucose
PubMed: 37773219
DOI: 10.1007/s00253-023-12797-6 -
Cancer Control : Journal of the Moffitt... 2021In the 1920s, Otto Warburg observed the phenomenon of altered glucose metabolism in cancer cells. Although the initial hypothesis suggested that the alteration resulted... (Review)
Review
In the 1920s, Otto Warburg observed the phenomenon of altered glucose metabolism in cancer cells. Although the initial hypothesis suggested that the alteration resulted from mitochondrial damage, multiple studies of the subject revealed a precise, multistage process rather than a random pattern. The phenomenon of aerobic glycolysis emerges not only from mitochondrial abnormalities common in cancer cells, but also results from metabolic reprogramming beneficial for their sustenance. The Warburg effect enables metabolic adaptation of cancer cells to grow and proliferate, simultaneously enabling their survival in hypoxic conditions. Altered glucose metabolism of cancer cells includes, inter alia, qualitative and quantitative changes within glucose transporters, enzymes of the glycolytic pathway, such as hexokinases and pyruvate kinase, hypoxia-inducible factor, monocarboxylate transporters, and lactate dehydrogenase. This review summarizes the current state of knowledge regarding inhibitors of cancer glucose metabolism with a focus on their clinical potential. The altered metabolic phenotype of cancer cells allows for targeting of specific mechanisms, which might improve conventional methods in anti-cancer therapy. However, several problems such as drug bioavailability, specificity, toxicity, the plasticity of cancer cells, and heterogeneity of cells in tumors have to be overcome when designing therapies based on compounds targeted in cancer cell energy metabolism.
Topics: Antineoplastic Agents; Glycolysis; Humans; Hypoxia-Inducible Factor 1; L-Lactate Dehydrogenase; Monocarboxylic Acid Transporters; Neoplasms; Warburg Effect, Oncologic
PubMed: 34554006
DOI: 10.1177/10732748211041243 -
Methods in Molecular Biology (Clifton,... 2020Apicomplexans are obligate parasites that replicate inside host cells, within a subcellular compartment called the parasitophorous vacuole. Egress is the process by...
Apicomplexans are obligate parasites that replicate inside host cells, within a subcellular compartment called the parasitophorous vacuole. Egress is the process by which apicomplexan parasites like Toxoplasma gondii exit from host cells, rupturing the parasitophorous vacuole and host-cell plasma membranes in the process. T. gondii retains the ability to egress throughout most of its intracellular replicative cycle, and this process has been associated with parasite signaling pathways that include the modulation of intracellular calcium, cyclic nucleotides, phosphatidic acid, and pH, which can be manipulated genetically or pharmacologically. Here we describe two methods of assessing stimulated parasite egress from host cells by measuring the permeabilization of host-cell membranes that occurs during this process. The first method measures the release of lactate dehydrogenase (LDH) from host cells, which is quantified in a colorimetric assay that detects LDH by the enzymatic generation of red formazan. The second method measures entry of the cell-impermeant 4',6-diamidino-2-phenylindole (DAPI) DNA dye, which stains host-cell nuclei (HCN) as parasites egress. Both described methods complement, with higher throughput, video-microscopy approaches that are well suited to examine the dissociation of parasite vacuoles that follows host-cell permeabilization.
Topics: Cell Nucleus; Colorimetry; Kinetics; L-Lactate Dehydrogenase; Toxoplasma
PubMed: 31758453
DOI: 10.1007/978-1-4939-9857-9_10 -
Biomedicine & Pharmacotherapy =... Mar 2022Cancer is one of the main causes of human mortality and brain tumors, including invasive pituitary adenomas, medulloblastomas and glioblastomas are common brain... (Review)
Review
Cancer is one of the main causes of human mortality and brain tumors, including invasive pituitary adenomas, medulloblastomas and glioblastomas are common brain malignancies with poor prognosis. Therefore, the development of innovative management strategies for refractory cancers and brain tumors is important. In states of mitochondrial dysfunction - commonly encountered in malignant cells - cells mostly shift to anaerobic glycolysis by increasing the expression of LDHA (Lactate Dehydrogenase-A) gene. Oxamate, an isosteric form of pyruvate, blocks LDHA activity by competing with pyruvate. By blocking LDHA, it inhibits protumorigenic cascades and also induces ROS (reactive oxygen species)-induced mitochondrial apoptosis of cancer cells. In preclinical studies, oxamate blocked the growth of invasive pituitary adenomas, medulloblastomas and glioblastomas. Oxamate also increases temozolomide and radiotherapy sensitivity of glioblastomas. Oxamate is highly polar, which may preclude its clinical utilization due to low penetrance through cell membranes. However, this obstacle could be overcome with nanoliposomes. Moreover, different oxamate analogs were developed which inhibit LDHC4, an enzyme also involved in cancer progression and germ cell physiology. Lastly, phenformin, an antidiabetic agent, exerts anticancer effects via complex I inhibition in the mitochondria and leading the overproduction of ROS. Oxamate combination with phenformin reduces the lactic acidosis-causing side effect of phenformin while inducing synergistic anticancer efficacy. In sum, oxamate as a single agent and more efficiently with phenformin has high potential to slow the progression of aggressive cancers with special emphasis to brain tumors.
Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Glycolysis; Humans; L-Lactate Dehydrogenase; Mitochondria; Neoplasms; Oxamic Acid; Phenformin; Radiation Tolerance; Reactive Oxygen Species; Temozolomide
PubMed: 35124385
DOI: 10.1016/j.biopha.2022.112686