Discovery of small molecules interacting at lactate dehydrogenases tetrameric interface using a biophysical screening cascade.

Lactate dehydrogenases (LDHs) are tetrameric enzymes of therapeutic relevance for cancer therapy due to their important implications in cancer cell metabolism. LDH active site inhibition suffers from different drawbacks due to several features such as high cellular concentration and a shared active site among the dehydrogenase family. Conversely, targeting the LDH oligomeric state is an exciting strategy that could provide a suitable alternative to active-site inhibition. In the present study, we developed a biophysical screening cascade to probe the LDHs tetrameric interface. Using nanoscale differential fluorimetry (nanoDSF) as a primary screening method, we identified a series of hits that destabilize the tetrameric protein. From this primary screening, we validated selected hits using saturation transfer difference nuclear magnetic resonance (STD NMR) and microscale thermophoresis (MST) as a combination of orthogonal biophysical techniques. Finally, we characterized the validated hits and demonstrated that they specifically interact at the tetrameric interface of LDH-1 and LDH-5 and can inhibit the LDH tetramerization process. Overall, this work provides a convenient method for screening ligands at the LDH tetrameric interface and has identified promising hits suitable for further optimization. We believe that this biophysical screening cascade, especially the use of (nano)DSF, could be extended to other homomeric proteins.

Structure, Function, and Thermodynamics of Lactate Dehydrogenases from Humans and the Malaria Parasite P. falciparum.

Temperature adaptation is ubiquitous among all living organisms, yet the molecular basis for this process remains poorly understood. It can be assumed that for parasite-host systems, the same enzymes found in both organisms respond to the same selection factor (human body temperature) with similar structural changes. Herein, we report the existence of a reversible temperature-dependent structural transition for the glycolytic enzyme lactate dehydrogenase (LDH) from the malaria parasite Plasmodium falciparum (pfLDH) and human heart (hhLDH) occurring in the temperature range of human fever. This transition is observed for LDHs from psychrophiles, mesophiles, and moderate thermophiles in their operating temperature range. Thermodynamic analysis reveals unique thermodynamic signatures of the LDH-substrate complexes defining a specific temperature range to which human LDH is adapted and parasite LDH is not, despite their common mesophilic nature. The results of spectroscopic analysis combined with the available crystallographic data reveal the existence of an active center within pfLDH that imparts psychrophilic structural properties to the enzyme. This center consists of two pockets, one formed by the five amino acids (5AA insert) within the substrate specificity loop and the other by the active site, that mutually regulate one another in response to temperature and induce structural and functional changes in the Michaelis complex. Our findings pave the way toward a new strategy for malaria treatments and drug design using therapeutic agents that inactivate malarial LDH selectively at a specific temperature range of the cyclic malaria paroxysm.

R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/m6A/PFKP/LDHB axis.

R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N6-methyladenosine (m6A)/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34+ hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism.

Reduction in urinary oxalate excretion in mouse models of Primary Hyperoxaluria by RNA interference inhibition of liver lactate dehydrogenase activity.

The Primary Hyperoxaluria's (PH) are rare autosomal recessive disorders characterized by elevated oxalate production. PH patients suffer recurrent calcium oxalate kidney stone disease, and in severe cases end stage renal disease. Recent evidence has shown that RNA interference may be a suitable approach to reduce oxalate production in PH patients by knocking down key enzymes involved in hepatic oxalate synthesis. In the current study, wild type mice and mouse models of PH1 (AGT KO) and PH2 (GR KO) were treated with siRNA that targets hepatic LDHA. Although siRNA treatment substantially reduced urinary oxalate excretion [75%] in AGT KO animals, there was a relatively modest reduction [32%] in GR KO animals. Plasma and liver pyruvate levels significantly increased with siRNA treatment and liver organic acid analysis indicated significant changes in a number of glycolytic and TCA cycle metabolites, consistent with the known role of LDHA in metabolism. However, siRNA dosing data suggest that it may be possible to identify a dose that limits changes in liver organic acid levels, while maintaining a desired effect of reducing glyoxylate to oxalate synthesis. These results suggest that RNAi mediated reduction of hepatic LDHA may be an effective strategy to reduce oxalate synthesis in PH, and further analysis of its metabolic effects should be explored. Additional studies should also clarify in GR KO animals whether there are alternate enzymatic pathways in the liver to create oxalate and whether tissues other than liver contribute significantly to oxalate production.

Blocking LDHA glycolytic pathway sensitizes glioblastoma cells to radiation and temozolomide.

PURPOSE: Up-regulation of lactate dehydrogenase LDHA, is a frequent event in human malignancies and relate to poor postoperative outcome. In the current study we examined the hypothesis that LDHA and anaerobic glycolysis, may contribute to the resistance of glioblastoma to radiotherapy and to temozolomide. METHODS AND MATERIALS: The expression of LDH5 isoenzyme (fully encoded by the LDHA gene) was assessed in human glioblastoma tissues. Experimental in vitro studies involved the T98 and U87 glioblastoma cell lines. Their sensitivity to radiotherapy and to temozolomide, following silencing of LDHA gene or following exposure to the LDHA chemical inhibitor 'oxamate' and to the glycolysis inhibitor '2-deoxy-d-glucose' (2DG), was studied. RESULTS: Glioblastoma tissues showed strong cytoplasmic and nuclear LDH5 expression in 0-90% (median 20%) of the neoplastic cells. T98 and U87 cell lines showed that blocking glycolysis, either with LDHA gene silencing or exposure to oxamate (30 mM) and blockage of glycolysis with 2DG (500 µM), results in enhanced radiation sensitivity, an effect that was more robust in the T98 radioresistant cell line. Furthermore, all three glycolysis targeting methods, significantly sensitized both cell lines to Temozolomide. CONCLUSIONS: The current study provides evidence that a large subgroup of human glioblastomas are highly glycolytic, and that inhibitors of glycolysis, like LDHA targeting agents, may prove of therapeutic importance by enhancing the efficacy of radiotherapy and temozolomide against this lethal disease.

The miR-34a-LDHA axis regulates glucose metabolism and tumor growth in breast cancer.

Lactate dehydrogenase A (LDHA) is involved in a variety of cancers. The purpose of this study was to investigate the expression, prognostic roles and function of LDHA in breast cancer. We found that LDHA was upregulated in both breast cancer cell lines and clinical specimens using quantitative real-time PCR (qRT-PCR). Immunohistochemistry (IHC) analysis of tissue microarrays (TMAs) showed that high LDHA expression was associated with cell proliferation, metastasis and poor patient overall survival (OS) and disease free survival (DFS). Furthermore, we found that LDHA promoted glycolysis and cell proliferation in vitro and in vivo. We also performed luciferase reporter assays and found that LDHA was a direct target of miR-34a. Repression of LDHA by miR-34a suppressed glycolysis and cell proliferation in breast cancer cells in vitro. Our findings provide clues regarding the role of miR-34a as a tumor suppressor in breast cancer through the inhibition of LDHA both in vitro and in vivo. Targeting LDHA through miR-34a could be a potential therapeutic strategy in breast cancer.

Inhibitory effects of ionic liquids on the lactic dehydrogenase activity.

Ionic liquids (ILs) were widely used in scientific and industrial application and have been reported to possess potential toxicity to the environment and human health. The effects of six typical N-methylimidazolium-based ILs ([Cnmim]X, n=4, 6, 8; X=Br(-), Cl(-), BF4(-), CF3SO3(-)) on the lactic dehydrogenase (LDH) activity and the molecular interaction mechanism of ILs and the LDH were investigated with the aid of spectroscopic techniques. Experimental results showed that the LDH activity was inhibited in the presence of ILs. For the ILs with the same anion but different cations, their inhibitory ability on the LDH activity increased with increasing the alkyl chain length on the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of LDH with the addition of ILs. Both positive ΔH and ΔS suggested that hydrophobicity was the major driven force in the interaction process as expected.

Modulation of acid phosphatase and lactic dehydrogenase in hexachlorocyclohexane-induced hepatocarcinogenesis in mice.

The present study was designed to elucidate the involvement of acid phosphatase (ACP) in metastasis and lactate dehydrogenase (LDH) as an immediate compensatory alleviation mechanism for energy stress in liver lesions induced by hexachlorocyclohexane in Swiss mice. Animals were continuously exposed to hexachlorocyclohexane (500 ppm) for 2, 4, and 6 months. Neoplastic nodules and tumors developed after continuous exposure for 4 and 6 months, respectively. The distribution pattern of both enzymes markedly varied in neoplastic nodules and tumors. Intense ACP activity was more observed only in sinusoids and blood vessels of neoplastic nodule, whereas an overall increase in ACP activity was observed in the tumor. Noticeably, a significant decline in LDH activity was noted after 2 and 4 months of exposure, whereas LDH in a tumor region showed intense enzymatic activity. The role of acid phosphate in metastasis and LDH in oxidative stress during hepatocarcinogenesis induced by hexachlorocyclohexane has been discussed.

Chemical composition of three polysaccharides from Gynostemma pentaphyllum and their antioxidant activity in skeletal muscle of exercised mice.

PURPOSE: To investigate the effects of polysaccharide from Gynostemma pentaphyllum on antioxidant activity in skeletal muscle of mice exercised to exhaustion. METHODS: Three polysaccharide fractions were obtained from G. pentaphyllum polysaccharide (GPP) and termed GPP1-a, GPP2-b, and GPP3-a. Gas chromatography (GC) and infrared spectrum of the polysaccharides were determined. The fractions were orally administrated to mice once daily for 1 wk. The exercise time to exhaustion was assessed using a forced swim test of mice after a week. The glucose, creatine phosphokinase, and lactic dehydrogenase in serum; the activity of superoxide dismutase and glutathione peroxidase; and the levels of malondialdehyde (MDA) and glycogen in muscle were determined. RESULTS: The results of GC demonstrated that GPP1-a, GPP2-b, and GPP3-a were composed of different monosaccharides with distinct molar ratios. Infrared spectrum showed that the main typicals of GPP1-a and GPP2-b were ß-configuration and the main typical of GPP3-a was α-configuration. Among the 3 fractions of GPP, GPP1-a administration significantly prolonged exercise time to exhaustion of mice, increased glycogen level and some of antioxidant enzyme activities, and decreased MDA level in muscle. CONCLUSIONS: The mechanism by which GPP1-a prolonged exercise time to exhaustion in mice may be associated with scavenging reactive oxygen species excessively produced and further increasing glycogen levels in skeletal muscle.

[Effect of lemon essential oil on caries factors of Streptococcus sobrinus].

OBJECTIVE: To investigate the mechanism of lemon peel essential oil (LPE) on the cariogenicity of Streptococcus sobrinus (Ss). METHODS: LPE was extracted by the authors, and the minimum inhibition concentration (MIC) was measured by disc diffusion method. The LPE was used as the experimental group with concentrations ranging from 2.250 g/L to 0.281 g/L prepared with trypticase peptone yeast (TPY) culture medium, and TPY culture medium was used as the control group. Ss at the concentration of 10(8) CFU/ml was added to each group, and cultured for 6, 18, 24, 48 hours. Neson-Somogyi method was used to measure the content of reducing sugar, and glucosyltransferase (GTF) activity. The activity of lactate dehydrogenase (LDH) was measured by lactic acid and pyruvic acid continuous monitoring method. The content of water insoluble glucan (WIG) was measured by anthrone method, and the pH value of the culture solution was detected. The value of pH before the experiment and the time difference was alculated as ΔpH. RESULTS: At the same time point, the activity of GTF and LDH and the concentration of WIG and the value ΔpH decreased gradually with the increase of concentration of LPE. There were significant differences between each experimental group and control group (P < 0.01). The control group had the maximum value, GTF: (6.71 ± 0.61) mIU, LDH: (135.8 ± 1.7) U/L, WIG: (47.15 ± 5.12) mg/L, ΔpH: (2.67 ± 0.01). The highest drug concentration group had the minimum value: GTF: (0.39 ± 0.07) mIU, LDH: (95.0 ± 5.4) U/L, WIG: (2.44 ± 0.38) mg/L, ΔpH: (0.61 ± 0.01). CONCLUSIONS: The LPE below the MIC could still inhibit the GTF, LDH activity and lead to the decrease of WIG and the acid production.

Transient enzyme kinetics: graph-theoretic approach.

A graph-theoretic approach is shown to simplify the analysis of transient enzyme kinetics. The coefficients of the characteristic polynomial for kinetic equations are obtained by graphical construction of directed trees and sub-trees in the kinetic scheme. An example of kinetic schemes, providing a simple time-dependent analytical solution, is demonstrated. This example describes a substrate-inhibited enzymatic reaction and interprets the pH-dependent inhibition of the lactate dehydrogenase. It is shown that rapid equilibrium in some parts of the kinetic scheme can simplify the analysis. The enzyme pre-incubation with a product is shown to be characterized by the non-monotonous transient kinetics. This phenomenon is useful to estimate correctly the kinetic parameters. It is supposed that the lactate dehydrogenase substrate inhibition can be important for switching the glycolytic fluxes.