Inhibition Of Lactate Dehydrogenase Research Articles

Photo-oxidation and Photoreduction of Catechols by Chlorophyll Metabolites and Methylene Blue.

While plant-derived oxidants can protect cells from oxidative damage, limited research has examined the role of dietary chlorophyll. Photoreduction of ubiquinone by chlorophyll metabolites and red light has been reported in vitro and in animal models. Herein we examined photo-oxidation and photoreduction reactions of catechols, dopamine and hydrocaffeic acid. Photo-oxidation of dopamine by methylene blue and the chlorophyll metabolites pheophorbide A, chlorin e6 and sodium copper chlorophyllin was studied by monitoring aminochrome, the cyclized product of the dopamine o-quinone with its amine. Singlet oxygen scavengers including sodium azide, ascorbate and glutathione decreased aminochrome formation by methylene blue and pheophorbide A. Addition of EDTA, a tertiary amine electron donor, to the reaction of dopamine, photosensitizer and red light decreased aminochrome formation. Photoreduction of the dopamine o-quinone produced by mushroom tyrosinase was achieved by both methylene blue and pheophorbide A only when an electron donor was included. Due to limited solubility, photo-oxidation and photoreduction reactions by pheophorbide A required 5-7.5% dimethylformamide for optimal reactivity. Catalytic photoreduction of 2,3-dimethoxy-5-methyl-p-benzoquinone by methylene blue or pheophorbide A and tertiary amine electron donors was observed. Among the chlorophyll metabolites, pheophorbide A was more effective than chlorin e6 or sodium copper chlorophyllin in photo-oxidation of dopamine and photoreduction reactions. Singlet oxygen inhibited lactate dehydrogenase A activity, and higher molecular weight protein cross-links were observed on SDS-PAGE. Hydrocaffeic acid competed with lactate dehydrogenase A for reaction with singlet oxygen produced by methylene blue; however, no protection by hydrocaffeic acid (HCA) was observed when pheophorbide A was used. Cysteine modification of lactate dehydrogenase A by the o-quinone of hydrocaffeic acid was detected using a redox cycling stain. Inclusion of an electron donor decreased protein labeling.

Tumor acidity: From hallmark of cancer to target of treatment.

Tumor acidity is one of the cancer hallmarks and is associated with metabolic reprogramming and the use of glycolysis, which results in a high intracellular lactic acid concentration. Cancer cells avoid acid stress major by the activation and expression of proton and lactate transporters and exchangers and have an inverted pH gradient (extracellular and intracellular pHs are acid and alkaline, respectively). The shift in the tumor acid–base balance promotes proliferation, apoptosis avoidance, invasiveness, metastatic potential, aggressiveness, immune evasion, and treatment resistance. For example, weak-base chemotherapeutic agents may have a substantially reduced cellular uptake capacity due to “ion trapping”. Lactic acid negatively affects the functions of activated effector T cells, stimulates regulatory T cells, and promotes them to express programmed cell death receptor 1. On the other hand, the inversion of pH gradient could be a cancer weakness that will allow the development of new promising therapies, such as tumor-targeted pH-sensitive antibodies and pH-responsible nanoparticle conjugates with anticancer drugs. The regulation of tumor pH levels by pharmacological inhibition of pH-responsible proteins (monocarboxylate transporters, H+-ATPase, etc.) and lactate dehydrogenase A is also a promising anticancer strategy. Another idea is the oral or parenteral use of buffer systems, such as sodium bicarbonate, to neutralize tumor acidity. Buffering therapy does not counteract standard treatment methods and can be used in combination to increase effectiveness. However, the mechanisms of the anticancer effect of buffering therapy are still unclear, and more research is needed. We have attempted to summarize the basic knowledge about tumor acidity.

5-ALA Is a Potent Lactate Dehydrogenase Inhibitor but Not a Substrate: Implications for Cell Glycolysis and New Avenues in 5-ALA-Mediated Anticancer Action.

Simple SummaryIn the present work, we found that 5-ALA, a natural precursor of heme, can hinder cell glycolysis, which is the main path of energy production for most cancer cells. More specifically, we found that 5-ALA can block an enzyme involved in glycolysis, called lactate dehydrogenase (LDH). We found that 5-ALA has a potency of LDH inhibition comparable to other established LDH inhibitors, such as oxamate or tartronic acid. Nevertheless, 5-ALA has a high accumulation rate in cancers and specifically in the incurable brain cancer glioblastoma multiforme (GBM), which is an important advantage. In fact, because of its high specificity to GBM, 5-ALA is used in the clinic to accurately guide the resection of the tumours, through the light emission of its photoactive product protoporphyrin IX (PpIX). PpIX is the penultimate step in the heme production. Importantly, we show here that continuous administration of 5-ALA killed GBM cells according to their dependence on glycolysis. We additionally found that 20% of externally administered 5-ALA is engaged in the inhibition of LDH, as when LDH was pre-loaded by another inhibitor, tartronic acid, then the cell production of PpIX from 5-ALA was increased by 20%. Since PpIX is an important drug for photodynamic therapy of cancer (excitation by light of PpIX produces oxygen by-products that can kill cancer cells), we additionally discovered that preloading LDH with its inhibitor tartronic acid before performing 5-ALA PDT increases the cancer cell death by 15%.In a course of metabolic experiments, we determined that the addition of δ-aminolevulinic acid (5-ALA) to a panel of glioblastoma multiforme (GBM) cells caused a steep reduction in their glycolytic activity. This reduction was accompanied by a decrease in adenosine triphosphate (ATP) production from glycolysis. These results suggested that 5-ALA is an inhibitor of glycolysis; due to the structural similarity of 5-ALA to the established lactate dehydrogenase (LDH) inhibitors oxamate (OXM) and tartronate (TART), we initially investigated LDH inhibition by 5-ALA in silico. The modelling revealed that 5-ALA could indeed be a competitive inhibitor of LDH but not a substrate. These theoretical findings were corroborated by enzymatic and cell lysate assays in which 5-ALA was found to confer a potent LDH inhibition comparable to that of OXM and TART. We subsequently evaluated the effect of 5-ALA-induced glycolysis inhibition on the viability of GBM cells with diverse metabolic phenotypes. In the Warburg-type cell lines Ln18 and U87, incubation with 5-ALA elicited profound and irreversible cell death (90–98%) at 10 mM after merely 24 h. In T98G, however, which exhibited both high respiratory and glycolytic rates, LD95 was achieved after 72 h of incubation with 20 mM 5-ALA. We additionally examined the production of the 5-ALA photosensitive metadrug protoporphyrin IX (PpIX), with and without prior LDH inhibition by TART. These studies revealed that ~20% of the 5-ALA taken up by the cells was engaged in LDH inhibition. We subsequently performed 5-ALA photodynamic therapy (PDT) on Ln18 GBM cells, again with and without prior LDH inhibition with TART, and found a PDT outcome enhancement of ~15% upon LDH pre-inhibition. We expect our findings to have a profound impact on contemporary oncology, particularly for the treatment of otherwise incurable brain cancers such as GBM, where the specific accumulation of 5-ALA is very high compared to the surrounding normal tissue.

Transcriptional, chromatin, and metabolic landscapes of LDHA inhibitor-resistant pancreatic ductal adenocarcinoma.

Metabolic reprogramming, due in part to the overexpression of metabolic enzymes, is a key hallmark of cancer cells. Lactate dehydrogenase (LDHA), a metabolic enzyme that catalyzes the interconversion of lactate and pyruvate, is overexpressed in a wide variety of cancer types, including pancreatic ductal adenocarcinoma (PDAC). Furthermore, the genetic or pharmacological inhibition of LDHA suppresses cancer growth, demonstrating a cancer-promoting role for this enzyme. Therefore, several pharmacological LDHA inhibitors are being developed and tested as potential anti-cancer therapeutic agents. Because cancer cells are known to rapidly adapt and become resistant to anti-cancer therapies, in this study, we modeled the adaptation of cancer cells to LDHA inhibition. Using PDAC as a model system, we studied the molecular aspects of cells resistant to the competitive LDHA inhibitor sodium oxamate. We performed unbiased RNA-sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), and metabolomics analyses of parental and oxamate-resistant PDAC cells treated with and without oxamate to identify the transcriptional, chromatin, and metabolic landscapes of these cells. We found that oxamate-resistant PDAC cells were significantly different from parental cells at the levels of mRNA expression, chromatin accessibility, and metabolites. Additionally, an integrative analysis combining the RNA-seq and ATAC-seq datasets identified a subset of differentially expressed mRNAs that directly correlated with changes in chromatin accessibility. Finally, functional analysis of differentially expressed metabolic genes in parental and oxamate-resistant PDAC cells treated with and without oxamate, together with an integrative analysis of RNA-seq and metabolomics data, revealed changes in metabolic enzymes that might explain the changes in metabolite levels observed in these cells. Collectively, these studies identify the transcriptional, chromatin, and metabolic landscapes of LDHA inhibitor resistance in PDAC cells. Future functional studies related to these changes remain necessary to reveal the direct roles played by these changes in the development of LDHA inhibitor resistance and uncover approaches for more effective use of LDHA inhibitors in cancer therapy.

Lactate Dehydrogenase-Inhibitors Isolated from Ethyl Acetate Extract of Selaginella doederleinii by Using a Rapid Screening Method with Enzyme-Immobilized Magnetic Nanoparticles.

Lactate dehydrogenase (LDH) is one of the important enzyme systems for glycolysis and gluconeogenesis. It can catalyze the reduction and oxidation reaction between propionic acid and L-lactic acid, which is usually overexpressed in cancer cells. Therefore, inhibiting the activity of LDH is a promising way for the treatment of cancer. In this study, an effective method based on ligand fishing and ultra performance liquid chromatography-mass spectrum (UPLC-MS) was established to screen and identify active ingredients from Selaginella doederleinii with potential inhibitory activity for LDH. Firstly, LDH was immobilized on the magnetic nanoparticles (MNPs), three immobilization parameters including LDH concentration, immobilization time and pH were optimized by single factor and response surface methodology for maximum (max) immobilization yield. Then, a mixed model of galloflavin and chlorogenic acid (inhibitors and non-inhibitors of LDH) was used to verify the specificity of immobilized LDH ligand fishing, and the conditions of ligand fishing were further optimized. Finally, combined with UPLC-MS, immobilized LDH was used to simultaneously screen and identify potential LDH inhibitors from the ethyl acetate extract of Selaginella doederleinii. The prepared fishing material was comprehensively characterized by scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and fourier transform infrared spectrometer (FT-IR). The optimal immobilization conditions were obtained as LDH concentration of 0.7 mg/mL, pH value of 4.5, and immobilization time of 3.5 h. Under these conditions, the max immobilization yield was (3.79 ± 0.08) × 103 U/g. The specificity analysis showed that immobilized LDH could recognize and capture ligands, and the optimal ligand fishing conditions included that the incubation time was 30 min, the elution time was 20 min, and the concentration of methanol as eluent was 80%. Finally, two LDH inhibitors, amentoflavone and robustaflavone, were screened by immobilized LDH from the ethyl acetate extract of Selaginella doederleinii. The study provided a meaningful evidence for discovering the bioactive constituents in ethyl acetate extract of Selaginella doederleinii related to cancer treatment, and this ligand fishing method was feasible for screening enzyme inhibitors from similar complex mixtures.

Naturally Occurring 8ß,13ß-kaur-15-en-17-al and Anti-Malarial Activity from Podocarpus polystachyus Leaves.

Despite much interest and studies toward the genus Podocarpus, the anti-malarial evaluation of Podocarpus polystachyus’s phytoconstituents remains lacking. Herein, the phytoconstituents of P. polystachyus leaves and their anti-malarial effect against Plasmodium falciparum were investigated for the first time. One new natural product, 8ß,13ß-kaur-15-en-17-al (1), along with three known compounds, 8ß,13ß-kaur-15-en-17-ol (2) and 13ß-kaur-16-ene (3), and α-tocopherol hydroquinone (4) were isolated via HR-ESI-MS and NMR analyses. Compounds 1 and 2 inhibited P. falciparum growth at 12 and 52 µM of IC50, respectively. Their anti-malarial activity was associated with the in silico P. falciparum lactate dehydrogenase (PfLDH) inhibition. Molecular docking of ligands 1 and 2 with the putative target PfLDH revealed ~−2 kcal/mol of binding energies more negative than the control. Molecular dynamic simulations (100 ns) showed equal or smaller deviation values (RMSD, RMSF, Rg) and stronger interactions of PfLDH-1 and PfLDH-2 complexes via at least one consistent H-bond than the control. Additionally, a slightly increased PfLDH H-bond profile in their interactions improved the PfLDH dynamic and structural stabilities. Overall, this study supports the relevance of 1 and 2 as plasmodial growth inhibitors with their putative anti-PfLDH activity, which could be a potential scaffold for developing anti-malarial drugs.

Aloe emodin-conjugated sulfonyl hydrazones as novel type of antibacterial modulators against S. aureus 25923 through multifaceted synergistic effects

Aloe emodin-conjugated sulfonyl hydrazones were designed and synthesized as novel type of antibacterial modulators. Aloe emodin benzenesulfonyl hydrazone 5a (AEBH-5a) was preponderant for the treatment of S. aureus 25923 (MIC = 0.5 μg/mL) over norfloxacin and presented high selectivity between bacterial membranes and mammalian membranes. Especially, AEBH-5a could eliminate the formed biofilms and relieve the development of S. aureus 25923 resistance. The antibacterial mechanism of AEBH-5a from extracellularity to intracellularity illustrated that AEBH-5a could destroy bacterial membrane integrity, leading to the leakage of protein and nucleic acid. Besides, AEBH-5a could not only interact with DNA and induce oxidative stress but also inhibit lactate dehydrogenase (LDH) activity as well as render metabolic inactivation. In silico ADME studies prediction of AEBH-5a revealed a favorable bioavailability score and prominent drug-likeness profile. This research showed that the multifaceted synergistic effect initiated by aloe emodin-conjugated sulfonyl hydrazones is a reasonable and effective tactic to combat menacing bacterial infections.

Discovery of novel human lactate dehydrogenase inhibitors: Structure-based virtual screening studies and biological assessment

Most cancer cells switch their metabolism from mitochondrial oxidative phosphorylation to aerobic glycolysis to generate ATP and precursors for the biosynthesis of key macromolecules. The aerobic conversion of pyruvate to lactate, coupled to oxidation of the nicotinamide cofactor, is a primary hallmark of cancer and is catalyzed by lactate dehydrogenase (LDH), a central effector of this pathological reprogrammed metabolism. Hence, inhibition of LDH is a potential new promising therapeutic approach for cancer. In the search for new LDH inhibitors, we carried out a structure-based virtual screening campaign. Here, we report the identification of a novel specific LDH inhibitor, the pyridazine derivative 18 (RS6212), that exhibits potent anticancer activity within the micromolar range in multiple cancer cell lines and synergizes with complex I inhibition in the suppression of tumor growth. Altogether, our data support the conclusion that compound 18 deserves to be further investigated as a starting point for the development of LDH inhibitors and for novel anticancer strategies based on the targeting of key metabolic steps.

Development of a novel lactate dehydrogenase A inhibitor with potent antitumor activity and immune activation

Lactate accumulation in the tumor microenvironment was shown to be closely related to tumor growth and immune escape, and suppression of lactate production by inhibiting lactate dehydrogenase A (LDHA) has been pursued as a potential novel antitumor strategy. However, only a few potent LDHA inhibitors have been developed and most of them did not show potent antitumor effects in vivo. To this end, we designed new LDHA inhibitors and obtained a novel potent LDHA inhibitor, ML‐05. ML‐05 inhibited cellular lactate production and tumor cell proliferation, which was associated with inhibition of ATP production and induction of reactive oxygen species and G1 phase arrest. In a mouse B16F10 melanoma model, intratumoral injection of ML‐05 significantly reduced lactate production, inhibited tumor growth, and released antitumor immune response of T cell subsets (Th1 and GMZB+CD8 T cells) in the tumor microenvironment. Moreover, ML‐05 treatment combined with programmed cell death‐1 Ab or stimulator of interferon genes protein (STING) could sensitize the antitumor activity in B16F10 melanoma model. Collectively, we developed a novel potent LDHA inhibitor, ML‐05, that elicited profound antitumor activity when injected locally, and was associated with the activation of antitumor immunity. In addition, ML‐05 could sensitize immunotherapies, which suggests great translational value.

Abstract 3537: Pharmacologic inhibition of the glycolytic pathway improves response to immune checkpoint blockade

Abstract Preferential engagement in glycolysis is a hallmark of cancer cells and contributes to the progression and metastasis of many tumor types, including melanoma and triple-negative breast cancer (TNBC). Tumor reliance on glycolysis is emerging as a mechanism of resistance to immunotherapy, due in part to lactate-mediated immunosuppression and competition for glucose between effector T cells and tumor cells within the tumor microenvironment. Elevated serum lactate dehydrogenase (LDH) levels are associated with poor outcomes in cancer patients, and we observed that serum lactate and LDH levels correlate with primary tumor burden in mice. We recently demonstrated that genetic dampening of LDH subunit A in 4T1 TNBC results in improved and long-lasting anti-tumor responses to CTLA-4 blockade in mice. Therefore, we hypothesize that combining LDH inhibitors (LDHi) with CTLA-4 blockade will be an effective strategy to combat resistance to anti-CTLA-4 therapy. Since activated T cells rely on glycolysis, we first determined that glycolytic cancers overexpress LDH (compared with immune cells) by analyzing single-cell transcripts from patient melanoma biopsies. LDHA gene expression is significantly higher in malignant cells than infiltrating CD8+ T cells, and we replicated these findings at the protein level in whole cell lysate from B16-F10 melanoma and 4T1 TNBC tumor cells and tumor-antigen specific T cells in vitro. We show that LDHi reduces tumor lactate production and glucose consumption without inhibiting anti-tumor T-cell killing in vitro. The anti-tumor effect of LDH inhibition requires adaptive immunity, as daily treatment with LDHi results in reduced tumor burden in immunocompetent but not immune-deficient mice. Finally, we show that targeting LDH in combination with CTLA-4 blockade is more effective in slowing B16-F10 growth compared with CTLA-4 blockade alone, and that this combination promotes effector T cell activation while destabilizing regulatory T cell function. Citation Format: Svena Verma, Inna Serganova, Sadna Budhu, Lauren Dong, Myat Ko, Levi Mangarin, Taha Merghoub, Jedd Wolchok, Roberta Zappasodi. Pharmacologic inhibition of the glycolytic pathway improves response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3537.

Abstract 5426: Down-regulation of cancer metabolism enhances radiosensitivity by impairing the heat shock response

Abstract Therapy (radiation) resistance can, inter alia, be caused by an increased lactate dehydrogenase (LDH) activity, elevated concentrations of the oncometabolite lactate and an overexpression of anti-apoptotic heat shock proteins (HSPs). In this study we aimed to break radioresistance by targeting the cancer metabolism. We could show, for the first time, that the lactate metabolism and heat shock response are co-regulated in two cancer cell systems (murine B16F10 melanoma cells, human LS174T colorectal adenocarcinoma cells). A CRISPR/Cas9 induced double knockout of LDHA/B (LDH−/−) as well as a pharmacological inhibition of LDH by Oxamate or GNE-140 reduces tumor growth, ROS production and synthesis of different HSPs, including Hsp90, Hsp70 and Hsp27. Moreover, the membrane Hsp70 density was significantly reduced on tumor cells after inhibition of the LDH activity as a consequence of a down-regulated lipid metabolism affecting the Hsp70-anchoring, tumor-specific glycosphingolipid Gb3. Our results demonstrate that influencing cancer metabolism increases radiation sensitivity by an impairment of the stress response. Since presently available LDH inhibitors have a low stability and high toxicity in vivo, we are currently investigating more potent LDH inhibitors with a beneficial safety profile. In summary, targeting the lactate metabolism provides a promising strategy to improve radiosensitivity by impairing the stress response. Citation Format: Melissa Schwab, Katharina Thunborg, Omid Azimzadeh, Christine von Toerne, Maxim Shevtsov, Masa Zdralevic, Jacques Pouyssegur, Kathrin Renner, Marina Kreutz, Peter Vaupel, Gabriele Multhoff. Down-regulation of cancer metabolism enhances radiosensitivity by impairing the heat shock response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5426.

LINC01605 promotes aerobic glycolysis through lactate dehydrogenase A in triple-negative breast cancer.

Breast cancer is the most prevalent cancer diagnosed in women and the major malignancy that threatens women health, thus we explored the role of long noncoding RNA LINC01605 in triple‐negative breast cancer (TNBC). We collected tissue samples from TNBC patients and cultured breast cancer cells to detect LINC01605 levels by RT‐PCR. We then constructed LINC01605 knockdown and LINC01605 overexpressed TNBC cell lines, cell proliferation was measured by CCK‐8 and colony formation assays, cell migration and invasion were measured by Transwell assay, and aerobic glycolysis of cells was detected. Furthermore, a downstream target gene was found, and its role was confirmed by mouse allogeneic tumor formation. It discovered that LINC01605 expression was significantly increased in TNBC patients, and its high expression predicted a low survival prognosis for TNBC patients. Stable knockdown of LINC01605 remarkably inhibited cell proliferation, migration, and invasion, as well as aerobic glycolysis by inhibiting lactate dehydrogenase A in TNBC cell lines. Notably, knockdown of LINC01605 suppressed in vivo tumor formation and migration in TNBC transplanted mice. In conclusion, targeting long noncoding RNA LINC01605 might serve as a therapeutic candidate strategy to treat patients with TNBC.

Intracerebral transformation of pyruvate to lactate is deleterious for the neurological function after an experimental cardiac arrest in rabbits

Cardiac arrest is a very severe public health issue associated with dramatic neurological injury after the return of spontaneous circulation. During ischemia, neurons can use lactate as an alternative source of energy by oxidazing it into pyruvate via the lactate dehydrogenase (LDH). In the context of cardiac arrest, the neurological consequences of this phenomenon are however unknown. Our aim is to decipher the role of the cerebral lactate consumption on neurological injury after a cardiac arrest. Rabbits were resuscitated after 10 min of ventricular fibrillation and randomly received an intravenous infusion of lactate or pyruvate (10 mg/kg/min, Lact and Pyr groups, n = 6 for each), or oxamate, an inhibitor of LDH (37.5 mg/kg/min, Oxa group, n = 6). In a first set of experiment, we measured the arteriojugular concentrations differences in lactate oxygen and glucose, as well as cerebral extracellular concentration in lactate, pyruvate and glucose via microdialysis catheter during 4 h after cardiac arrest. Secondarly, we evaluated the neurological recovery of additional rabbits during 48 h after cardiac arrest. In both procedures, neuronal death and astrogliosis were assessed histologically. In control animals, cardiac arrest led to an intense metabolic crisis characterised by high cerebral consumptions in oxygen, glucose and lactate. Lactate administration slightly increased neurological dysfunction and neuronal death in comparison to control. Pyruvate administration led to a significant increase in the extracellular concentration in lactate 30 min after ROSC (8.4 ± 0.5 vs. 6.8 ± 1.1 fold change from baseline in Pyr and control groups respectively), which was associated with an aggravation of the neurological dysfunction, the number of degenerative neurons and astrogliosis. Inhibition of LDH by oxamate administration mitigated dramatically this metabolic crisis and was associated with improvement of neurological function and a decrease of neuronal death and astrogliosis compared to control group (15 ± 6 vs. 60 ± 9% of neurological dysfunction in Oxa and control groups, respectively). Our results state that cerebral oxidation of lactate into pyruvate seems to be related to neurological injury after cardiac arrest.

Tumor metabolite lactate promotes tumorigenesis by modulating MOESIN lactylation and enhancing TGF-β signaling in regulatory Tcells.

Regulatory T (Treg) cells play a vital role in maintaining the immunosuppressive tumor microenvironment. Lactate is a crucial metabolite in cancer and is related to tumor prognosis, metastasis, and overall survival. In this study, we focus on the effects of lactate on Treg cells. Invitro, lactate improves Treg cell stability and function, whereas lactate degradation reduces Treg cell induction, increases antitumor immunity, and decreases tumor growth in mice. Mechanistically, lactate modulates Treg cell generation through lactylation of Lys72 in MOESIN, which improves MOESIN interaction with transforming growth factor β (TGF-β) receptor I and downstream SMAD3 signaling. Cotreatment with anti-PD-1 and a lactate dehydrogenase inhibitor has a stronger antitumor effect than anti-PD-1 alone. Individuals with hepatocellular carcinoma who responded to anti-PD-1 treatment have lower levels of MOESIN lactylation in Treg cells than nonresponding individuals. Thus, we identify lactate as an essential small molecule that reinforces Treg cells in the tumor microenvironment through lactylation.

Abstract No. 301 Effect of transarterial embolization with lactate dehydrogenase inhibitor on target and non-target hepatocellular carcinoma tumors

Despite best practices, recurrence following transarterial chemoembolization is still common as hepatocellular carcinoma (HCC) cells undergo metabolic reprogramming to utilize anaerobic metabolic pathway via lactate dehydrogenase (LDH), enabling their survival. The goal of this project was to assess the growth response and rates of recurrence of HCC target and non-target (NT) tumors to transarterial embolization (TAE) with an LDH inhibitor (LDHi) as compared with bland TAE.

The Lactate and the Lactate Dehydrogenase in Inflammatory Diseases and Major Risk Factors in COVID-19 Patients.

—Lactate dehydrogenase (LDH) is a terminating enzyme in the metabolic pathway of anaerobic glycolysis with end product of lactate from glucose. The lactate formation is crucial in the metabolism of glucose when oxygen is in inadequate supply. Lactate can also be formed and utilised by different cell types under fully aerobic conditions. Blood LDH is the marker enzyme, which predicts mortality in many conditions such as ARDS, serious COVID-19 and cancer patients. Lactate plays a critical role in normal physiology of humans including an energy source, a signaling molecule and a pH regulator. Depending on the pH, lactate exists as the protonated acidic form (lactic acid) at low pH or as sodium salt (sodium lactate) at basic pH. Lactate can affect the immune system and act as a signaling molecule, which can provide a “danger” signal for life. Several reports provide evidence that the serum lactate represents a chemical marker of severity of disease similar to LDH under inflammatory conditions. Since the mortality rate is much higher among COVID-19 patients, associated with high serum LDH, this article is aimed to review the LDH as a therapeutic target and lactate as potential marker for monitoring treatment response of inflammatory diseases. Finally, the review summarises various LDH inhibitors, which offer potential applications as therapeutic agents for inflammatory diseases, associated with high blood LDH. Both blood LDH and blood lactate are suggested as risk factors for the mortality of patients in serious inflammatory diseases.

Allosteric Regulation of the Mobile Loop in Lactate Dehydrogenase from Plasmodium Falciparum

Malaria is still considered one of the deadliest parasitic diseases. Plasmodium parasites that cause malaria, particularly P. falciparum, have become resistant to many commonly used treatments. To combat this illness, we propose developing novel therapeutics that selectively target the mobile loop region of lactate dehydrogenase (LDH), an enzyme that the parasite depends on for survival. In general, LDH catalyzes the interconversion of pyruvate to lactate and NADH to NAD+. The mobile loop of LDH from P. falciparum (pfLDH) is located proximal to the active site and contains five additional residues compared to mammalian LDH (Figure 1). This significant structural change makes the mobile loop region of pfLDH an attractive target for the development of therapeutics, as it contains a catalytically necessary arginine residue that interacts with the substrate upon loop closure. While inhibitors of LDH, both small molecule and peptide‐based, have been previously identified and developed, it is unclear if the mobile loop region of LDH serves as a viable target for drug discovery.Preliminary data recently obtained demonstrates that a previously developed DNA aptamer (2008s), designed to bind to the mobile loop region of pfLDH, inhibits the enzyme selectively over mammalian LDH. The developers of 2008s reported that the aptamer does not affect the kinetics of the enzyme. However, our steady‐state kinetics measurements show that the kcat or turnover number of pfLDH is initially decreased by half while the Km or Michaelis constant remains about the same at the highest aptamer concentration studied (100 nM). The decrease in Kcat with constant Km is characteristic of an uncompetitive inhibitor that does not compete with the substrate for the active site but decreases the overall rate of the enzyme reaction. The kinetic traces for these assays also show that activity fully recovers after approximately 20‐30 seconds, compared to assays performed in the absence of aptamer. Together, these preliminary data suggest that 2008s binds selectively to the pfLDH and inhibit the ternary complex initially; however, once the enzyme begins to turn over, the aptamer dissociates from the enzyme.Our results provide proof of concept that molecules designed to bind to the mobile loop of pfLDH can inhibit the enzyme. MD simulations using GROMACS will be utilized to explore the conformation space of the pfLDH loop motion and compare it to that of hhLDH. Regarding pfLDH, sampling the conformational space of the loop motion will allow for a better understanding of how aptamer binding impacts its dynamics and may also elucidate a druggable site on or near the mobile loop. Additional kinetics studies will be performed at higher aptamer concentrations to determine if more pronounced inhibitory effects are achievable. The development of allosteric inhibitors that bind to and limit the dynamics of the mobile loop region of pfLDH selectively could lead to a paradigm shift concerning the design of novel therapeutics and treatments for malaria and other diseases.

New salicylic acid derivatives, double inhibitors of glycolate oxidase and lactate dehydrogenase, as effective agents decreasing oxalate production

The synthesis and biological evaluation of double glycolate oxidase/lactate dehydrogenase inhibitors containing a salicylic acid moiety is described. The target compounds are obtained in an easily scalable two-step synthetic procedure. These compounds showed low micromolar IC50 values against the two key enzymes in the metabolism of glyoxylate. Mechanistically they behave as noncompetitive inhibitors against both enzymes and this fact is supported by docking studies. The biological evaluation also includes in vitro and in vivo assays in hyperoxaluric mice. The compounds are active against the three types of primary hyperoxalurias. Also, possible causes of adverse effects, such as cyclooxygenase inhibition or renal toxicity, have been studied and discarded. Altogether, this makes this chemotype with drug-like structure a good candidate for the treatment of primary hyperoxalurias.

Targeting pancreatic cancer with combinatorial treatment of CPI-613 and inhibitors of lactate metabolism.

Pancreatic cancer is the fourth leading cause of cancer death, with a 5-year survival rate of 10%. A stagnant high mortality rate over the last decades highlights the need for innovative therapeutic approaches. Pancreatic tumors pursue an altered metabolism in order to maintain energy generation under low nutrient influx and hypoxic conditions. Targeting these metabolic strategies might therefore be a reasonable therapeutic approach for pancreatic cancer. One promising agent is CPI- 613, a potent inhibitor of two enzymes of the tricarboxylic acid cycle. The present study evaluated the anti-cancerous efficacy of CPI-613 in combination with galloflavin, a lactate dehydrogenase inhibitor or with alpha-cyano-4-hydroxycinnamic acid, an inhibitor of monocarboxylate transporters. The efficacy of both combination therapies was tested in vitro on one human and two murine pancreatic cancer cell lines and in vivo in an orthotopic pancreatic cancer model. Tumor progression was evaluated by MRI and 18F-FDG PET-CT. Both combinatorial treatments demonstrated in vitro a significant inhibition of pancreatic cancer cell proliferation and induction of cell death. In contrast to the in vitro results, both combination therapies did not significantly reduce tumor growth in vivo. The in vitro results suggest that a combined inhibition of different metabolic pathways might be a promising approach for cancer therapy. However, the in vivo experiments indicate that applying a higher dosage or using other drugs targeting these metabolic pathways might be more promising.

Lactate Dehydrogenase as a Potential Therapeutic Drug Target to Control Babesia bigemina

Babesia bigemina is a tick-borne apicomplexan hemoprotozoan responsible for bovine babesiosis. The current drugs used for bovine babesiosis treatment have several drawbacks, including toxicity, the lack of effectiveness to clear the parasite, and potential to develop resistance. Identifying compounds that target essential and unique parasite metabolic pathways is a rational approach toward finding alternative drug treatments. Based on the genome sequence and transcriptomics analysis, it can be inferred that anaerobic glycolysis is the dominant adenosine triphosphate (ATP) supply for Babesia, and lactate dehydrogenase (LDH) is one of the essential enzymes in this pathway. Furthermore, the Babesia LDH sequence is distinct from its bovine homologue and thus a potential chemotherapeutic target that would result in decreasing the ATP supply to the parasite but not to the host. Gossypol is a known efficient specific inhibitor of LDH in the sensu stricto B. bovis and the sensu lato B. microti, among other related parasites, but no such data are currently available in the sensu stricto B. bigemina parasites. Hereby, we show that the LDH amino acid sequence is highly conserved among sensu stricto but not in sensu lato Babesia spp. A predictive structural analysis of B. bigemina LDH showed the conservation of the key amino acids involved in the binding to gossypol compared to B. bovis. Gossypol has a significant (P < 0.0001) inhibitory effect on the in vitro growth of B. bigemina, with IC50 of 43.97 mM after 72 h of treatment. The maximum IC (IC98) was observed at 60 mM gossypol. However, a significant effect on the viability of cattle PBMC was observed when the cells were cultured with 60 mM (IC98) gossypol compared with DMSO-exposed control cells. Interestingly, B. bigemina cultured at 3% oxygen expresses significantly higher levels of LDH and is more resistant to gossypol than the parasites maintained at ambient conditions containing ~20% oxygen. Altogether, the results suggest the potential of gossypol as an effective drug against B. bigemina infection, but the risk of host toxicity at therapeutic doses should be further evaluated in in vivo studies.