Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH).

Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease with a 5-year survival rate of merely 8%. The tumor microenvironment of PDAC is one of the factors contributing to drug resistance. More specifically, the hypoxic tumor core and the metabolic switch to aerobic glycolysis (the Warburg effect), contribute to the lack of drug response. Therefore, we investigated the effect of several novel lactate dehydrogenase (LDH-A) inhibitors (N-Hydroxyindole-based LDH-A inhibitors, NHI-1 and NHI-2, and the glucose conjugate NHI-Glc-2) in PDAC cells in vitro and in vivo, in combination with the standard drug gemcitabine. For this purpose we used our primary PDAC cancer cell cultures, tested growth inhibition with the SRB chemosensitivity assay, used 3D cultures and established an in vivo orthotopic bioluminescent model. Additionally, LDH-A enzyme activity inhibition by NHI-Glc-2 was assessed by spectrophotometry. LDH-A is overexpressed in PDAC and its expression is correlated with the prognosis of metastatic PDAC. The glucose transporter 1 (GLUT-1) is also overexpressed in PDAC, which would enable an increased uptake of NHI-Glc-2 by the tumor cells. LDH-A mRNA expression and enzyme activity were about 2-fold higher under hypoxic conditions. NHI-1, NHI-2 and NHI-Glc-2 were 4-15-fold more effective under hypoxic conditions compared to normoxia, but gemcitabine was 10-20-fold less active under hypoxia. NHI-1 showed a synergistic effect with gemcitabine in hypoxic PANC-1 and LPC006 cells (combination index 0.14 ± 0.06 and 0.29 ± 0.53, respectively). NHI-Glc-2 inhibited PDAC cell growth in micromolar range under hypoxic conditions and also showed a synergistic effect with gemcitabine. In a 3D spheroid culture (with a hypoxic core), NHI-Glc-2 disrupted the spheroid integrity. Moreover, in an orthotopic PDAC model NHI-Glc-2 showed a more pronounced inhibition (almost complete) of tumor growth compared to gemcitabine. NHI-Glc-2 also showed a favorable pharmacokinetics with a peak plasma concentration of 26 µM at 4 hr, which is higher than the IC50. In conclusion, LDH-A is a viable target in PDAC, and novel LDH-A inhibitors offer an innovative therapeutic tool. Remarkably, the LDH-A inhibitors NHI-1 and NHI-2 increased the effect of gemcitabine under hypoxic conditions, while the glucose conjugated NHI-Glc-2 showed an improved uptake possibly because of the increased GLUT-1 expression, leading to a pronounced in vivo effect.Citation Format: Btissame El Hassouni, Rocco Sciarrillo, Valentina Edith Gómez, Mina Maftouh, Giulia Mantini, Christian M. Vonk, Carlotta Granchi, Niccola Funel, Filippo Minutolo, Godefridus J. Peters, Elisa Giovannetti. Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3082.

<div>Abstract<p>Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both <i>in vitro</i> and <i>in vivo</i> by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis.</p>Significance:<p>LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.</p></div>

The aim of this study was to evaluate the effects and molecular mechanisms of everolimus on Panc-1 human pancreatic cancer cells. Panc-1 human pancreatic cancer cells were treated with everolimus (10 μg/ml) at selected time points (6, 12 and 24 h). Cell proliferation and apoptosis were evaluated by MTT and flow cytometric analyses. The glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. The activity of mammalian target of rapamycin (mTOR) signaling was measured by western blotting. The expression of genes, including hexokinase 2 (HK2) and microRNA-143 (miR-143), was evaluated by real-time polymerase chain reaction (PCR). The administration of everolimus time-dependently inhibited proliferation and glycolysis and induced apoptosis in the Panc-1 human pancreatic cancer cells. As the time of treatment with everolimus increased, the mTOR signaling activity decreased, indicated by lower phosphorylation levels of S6 kinase; however, the phosphorylation levels of mTOR barely changed. Moreover, our data showed an everolimus-induced increase in miR-143 and decrease in HK2 in Panc-1 cells in a time-dependent manner. In conclusion, the current study indicates a novel role of everolimus in its antitumor effect as an inhibitor of glycolysis in Panc-1 human pancreatic cancer cells. Furthermore, our data highlights the significance of exploring the mechanisms of everolimus and miR-143 in malignant tumors.

The abnormal metabolism of cancer cells is a crucial feature of tumors and provides promising therapeutic targets for cancer treatments. Aerobic glycolysis in cancer cells, termed the Warburg effect, is a highlighted characteristic of cancer‑specific metabolism. However, the effect of glycolysis inhibition on hepatocarcinogenesis remains to be elucidated. In the present study, the effects of the glycolysis inhibitor 2‑deoxy‑D‑glucose (2‑DG) on the N‑diethylnitrosamine (DEN)‑induced rat hepatocarcinoma model and its underlying mechanisms were investigated. It was observed that 2‑DG significantly delayed hepatocarcinogenesis and effectively prolonged survival time in the DEN‑treated rats. The glycolysis inhibitor, 2‑DG prominently decreased cell proliferation and increased cell apoptosis in the DEN‑induced rat hepatoma and had no evident impact on the pericarcinomatous liver tissues. Further investigation revealed that 2‑DG resulted in a reduction of glycolysis products, the compensatory increase of hexokinase 2 expression and a decrease in 6‑phosphofructo‑2‑kinase, pyruvate kinase M2 and lactate dehydrogenase A expression in the hepatoma tissues. The inhibition of glycolysis further suppressed the tricarboxylic acid cycle, fatty acid and cholesterol biosynthesis and ATP production, while it promoted autophagic activation. In addition, the in vitro study demonstrated that hypoxia, an important factor in the tumor microenvironment, may assist in increasing 2‑DG‑induced inhibition of cell viability, cell cycle retardation and the decrease of colony formation ability in hepatoma cells. Taken together, the present results suggested that 2‑DG may inhibit hepatocarcinogenesis in the DEN‑treated rats via restricting cancer cell metabolism. This finding provides a promising measure in the prevention and treatment of hepatoma.

Dynamic Imaging of LDH Inhibition in Tumors Reveals Rapid in vivo Metabolic Rewiring and Vulnerability to Combination Therapy

Reprogramming cancer metabolism has become the hallmark of cancer progression. As the key enzyme catalyzing the conversion of pyruvate to lactate in aerobic glycolysis of cancer cells, human lactate dehydrogenase (LDH) has been a promising target in the discovery of anticancer agents. Natural products are important sources of new drugs. Up to now, some natural compounds have been reported with the activity to target LDH. To give more information on the development of LDH inhibitors and application of natural products, herein, we reviewed the natural compounds with inhibition of LDH from diverse structures and discussed the future direction of the discovery of natural LDH inhibitors for cancer therapy.

Inhibitors of human lactate dehydrogenase (hLDH5)—the enzyme responsible for the conversion of pyruvate to lactate coupled with oxidation of NADH to NAD+—are promising therapeutic agents against cancer because this enzyme is generally found to be overexpressed in most invasive cancer cells and is linked to their vitality especially under hypoxic conditions. Consequently, significant efforts have been made for the identification of small-molecule hLDH5 inhibitors displaying high inhibitory potencies. X-ray structure of hLDH5 complexes as well as molecular modeling studies contribute to identify and explain the main binding modes of hLDH5 inhibitors reported in literature. The purpose of this review is to analyze the main three-dimensional interactions between some of the most potent inhibitors and hLDH5, in order to provide useful suggestions for the design of new derivatives.

The basic synthetic methods for obtaining N-hydroxyindoles were studied. The first method is intramolecular reductive cyclization of ortho-substituted nitroaromatic compounds by various reducing agents. Nitrophenyl acetaldehydes (synthesis by Acheson), N,N-disubstituted amino-2-nitrostyrenes (synthesis by Somei), ortho-nitroketoetheres of different structure or 1-(o-nitroarene)-1-cyanoalkylnitroketones, ortho-nitrophenylacetonitriles were used as substrates for the synthesis of N-hydroxyindoles. Titanium chloride (III), zinc in THF solution of ammonium chloride or in acetic acid, stannous chloride in ethanolic HCl, and hydrogen with application of catalytic agent on basis of noble metals (platinum, palladium) were used as reduction agents. The second method is a [3+2]-cycloaddition with nitrozoaromatic compounds to alkynes or deoxygenation ortho-nitro substituted aromatic compounds with subsequent intramolecular cyclization. The third method is catalytic oxidation of 2,3-dihydroindoles or different indoles by concentrated hydrogen peroxide with application of of tungstate sodium as catalyst. We attended special attention to synthesis of functional derivatives of N-hydroxyindole-5,6-dicarboxylic acids because these compounds were synthesized from commercially available basic materials using well known synthetic methods and without using expensive catalysts. Indoles of various designs were synthesized. They may be building blocks for the synthesis of biologically active substances, phthalocyanines of different structures having mesomorphic and catalytic properties. Compounds of this class of heterocycles were found among many natural products and they can be used as therapeutic agents, for example, as inhibitors of human lactate dehydrogenase of isoforms 5, selective inhibitors of monoamine oxidase A and B, and N-methoxyindole can be used to treat breast cancer. For citation:Chirkova Zh.V. Synthesis of N-hydroxyindoles. Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol. 2017. V. 60. N 4. P. 4-20.

The effects of Cu2+ on glycolysis and several glycolytic enzymes were studied in rat brain extracts in vitro. At concentrations reportedly found in Wilson's disease, Cu2+ significantly inhibited lactate production from glucose or glucose-6-phosphate in rat brain postnuclear supernatant with an IC50 of about 3 microM. Cu2+ also inhibited several glycolytic enzymes. Amongst the latter, Cu2+ was most effective in inhibiting hexokinase (IC50 for Cu2+ = 7 microM), moderately effective in inhibiting pyruvate kinase (IC50 for Cu2+ = 56 microM), but least effective in inhibiting lactate dehydrogenase (IC50 for Cu2+ = 300 microM). These results suggest that inhibition of brain glycolysis may have pathophysiological importance in copper poisoning and in Wilson's disease.

The switch from oxidative phosphorylation to glycolytic metabolism results in cells that generate fewer reactive oxygen species (ROS) and are resistant to the intrinsic induction of apoptosis. As a consequence, glycolytic cancer cells are resistant to radiation and chemotherapeutic agents that rely on production of ROS or intrinsic apoptosis. Further, the level of glycolysis correlates with tumor invasion, making glycolytic cancer cells an important target for new therapy development. We have synthesized a novel redox-active quinone phloroglucinol derivative, PMT7. Toxicity of PMT7 was in part due to loss of mitochondrial membrane potential in treated cells with subsequent loss of mitochondrial metabolic activity. Mitochondrial gene knockout ρ0 cells, a model of highly glycolytic cancers, were only half as sensitive as the corresponding wild-type cells and metabolic pathways downstream of MET were unaffected in ρ0 cells. However, PMT7 toxicity was also due to a block in autophagy. Both wild-type and ρ0 cells were susceptible to autophagy blockade, and the resistance of ρ0 cells to PMT7 could be overcome by serum deprivation, a situation where autophagy becomes necessary for survival. The stress response class III deacetylase SIRT1 was not significantly involved in PMT7 toxicity, suggesting that unlike other chemotherapeutic drugs, SIRT1-mediated stress and survival responses were not induced by PMT7. The dependence on autophagy or other scavenging pathways makes glycolytic cancer cells vulnerable. This can be exploited by induction of energetic stress to specifically sensitize glycolytic cells to other stresses such as nutrient deprivation or potentially chemotherapy.

Structure-based optimization of hydroxylactam as potent, cell-active inhibitors of lactate dehydrogenase

Lactate dehydrogenase (LDH) from Schistosoma mansoni has peculiar properties for a eukaryotic LDH. Schistosomal LDH (SmLDH) isolated from schistosomes, and the recombinantly expressed protein, are strongly inhibited by ATP, which is neutralized by fructose-1,6-bisphosphate (FBP). In the conserved FBP/anion binding site we identified two residues in SmLDH (Val187 and Tyr190) that differ from the conserved residues in LDHs of other eukaryotes, but are identical to conserved residues in FBP-sensitive prokaryotic LDHs. Three-dimensional (3D) models were generated to compare the structure of SmLDH with other LDHs. These models indicated that residues Val187, and especially Tyr190, play a crucial role in the interaction of FBP with the anion pocket of SmLDH. These 3D models of SmLDH are also consistent with a competitive model of SmLDH inhibition in which ATP (inhibitor) and FBP (activator) compete for binding in a well-defined anion pocket. The model of bound ATP predicts a distortion of the nearby key catalytic residue His195, resulting in enzyme inhibition. To investigate a possible physiological role of this allosteric regulation of LDH in schistosomes we made a kinetic model in which the allosteric regulation of the glycolytic enzymes can be varied. The model showed that inhibition of LDH by ATP prevents fermentation to lactate in the free-living stages in water and ensures complete oxidation via the Krebs cycle of the endogenous glycogen reserves. This mechanism of allosteric inhibition by ATP prevents the untimely depletion of these glycogen reserves, the only fuel of the free-living cercariae. Neutralization by FBP of this ATP inhibition of LDH prevents accumulation of glycolytic intermediates when S. mansoni schistosomula are confronted with the sudden large increase in glucose availability upon penetration of the final host. It appears that the LDH of S. mansoni is special and well suited to deal with the variations in glucose availability the parasite encounters during its life cycle.

Reprogramming of cell metabolism is essential for tumor progression and the best-studied metabolic phenomenon of cancer cells is aerobic glycolysis, in which pyruvate kinase isozyme type M2 (PKM2) plays a critical role. Follicle-stimulating hormone (FSH) contributes to epithelial ovarian cancer progression and has been shown to regulate cell metabolism in ovaries. The aim of this study was to investigate the interaction between FSH and PKM2 and their effect on aerobic glycolysis and cell proliferation in ovarian cancer. SKOV3 and OVCAR3 ovarian cancer cells were treated with FSH at various doses to investigate its effect on cell proliferation and PKM2 expression. siRNA-PKM2-transfected SKOV3 and OVCAR3 cells were treated with FSH to examine whether the changes induced by FSH could be altered by siRNA-PKM2. Glucose and lactate levels were evaluated to observe the change in glycolysis in these cells. In the current study, FSH upregulated the expression of PKM2 and glycolysis in SKOV3 and OVCAR3 cells. PKM2 knockdown reduced FSH-induced cell growth and glycolysis. Moreover, FSH attenuated apoptosis that was induced by the inhibition of PKM2. Collectively, the findings of this study indicated that FSH promoted glycolysis in epithelial ovarian cancer cells. Knockdown of PKM2 inhibited aerobic glycolysis and cell proliferation induced by FSH.

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.

Anticancer effects of glucosamine, a glucose derivative with a similar structure to glucose, have been discovered, but the molecular mechanisms are not yet fully understood. Since glucosamine and glucose not only have similar structures but also are catalyzed by the same enzyme, hexokinase (HK), in this study, we delved into determining whether the anticancer effect of glucosamine involves the inhibition of glycolysis by competition with glucose. Whole-genome screening analysis showed that many of the gene pathways controlled by glucosamine were directly and indirectly involved in glycolysis. In vitro experiments revealed that as more glucose was added, the anticancer effect of glucosamine decreased. Also, we found that glucosamine was transported into cells mainly through glucose transporter (GLUT) 2 which was responsible for the anticancer effects of glucosamine. In addition, we found that cancer cells with low expression level of HKII show high sensitivity to glucosamine, and a HK inhibitor, 3-bromopyruvate, enhanced the anticancer effect of glucosamine. Under hypoxic conditions, activated hypoxia-inducible factor 1α (HIF-1α) inducing glucose uptake and glycolysis hampered glucosamine-induced cell death, and HIF1A knockdown or HK inhibitors restored glucosamine’s anticancer effects. These findings demonstrate that glucosamine is an efficient glycolysis inhibitor and that GLUT2 and HKII play important roles as biomarkers for determining sensitivity to glucosamine. Moreover, our results suggest that the anticancer effect of glucosamine may be more efficient when administered in combination with other glycolytic agents or inhibitors targeting HIF-1α. Citation Format: Se Yong Park, Ki-Hoon Song, Ju-Hee Kang, Jung Ho Hwang, Seung Hyun Oh. Glucose transporter 2-transported glucosamine inhibits glycolysis in cancer cells through competition with glucose for hexokinase 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4170.