3-Bromopyruvate: A new strategy for inhibition of glycolytic enzymes in Leishmania amazonensis.

The compound 3-bromopyruvate (3-BrPA) is well-known and studies from several researchers have demonstrated its involvement in tumorigenesis. It is an analogue of pyruvic acid that inhibits ATP synthesis by inhibiting enzymes from the glycolytic pathway and oxidative phosphorylation. In this work, we investigated the effect of 3-BrPA on energy metabolism of L. amazonensis. In order to verify the effect of 3-BrPA on L. amazonensis glycolysis, we measured the activity level of three glycolytic enzymes located at different points of the pathway: (i) glucose kinases, step 1, (ii) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), step 6, and (iii) enolase, step 9. 3-BrPA, in a dose-dependent manner, significantly reduced the activity levels of all the enzymes. In addition, 3-BrPA treatment led to a reduction in the levels of phosphofruto-1-kinase (PFK) protein, suggesting that the mode of action of 3-BrPA involves the downregulation of some glycolytic enzymes. Measurement of ATP levels in promastigotes of L. amazonensis showed a significant reduction in ATP generation. The O2 consumption was also significantly inhibited in promastigotes, confirming the energy depletion effect of 3-BrPA. When 3-BrPA was added to the cells at the beginning of growth cycle, it significantly inhibited L. amazonensis proliferation in a dose-dependent manner. Furthermore, the ability to infect macrophages was reduced by approximately 50% when promastigotes were treated with 3-BrPA. Taken together, these studies corroborate with previous reports which suggest 3-BrPA as a potential drug against pathogenic microorganisms that are reliant on glucose catabolism for ATP supply.

Inhibition of energy metabolism by 3-bromopyruvate in the hard tick Rhipicephalus microplus.

The cattle tick R. microplus is the biggest obstacle to livestock rearing in tropical countries. It is responsible for billions of dollars in losses every year, affecting meat and milk production, beef and dairy cattle, and the leather industry. The lack of knowledge and strategies to combat the tick only increases the losses, it leads to successive and uncontrolled applications of acaricides, favouring the selection of strains resistant to commercially available chemical treatments. In this paper, we tested 3­bromopyruvate (3­BrPA), an alkylating agent with a high affinity for cysteine residues, on the R. microplus metabolism. We found that 3-BrPA was able to induce cell death in an assay using BME26 strain cell cultures derived from embryos, it was also able to reduce cellular respiration in developing embryos. 3-BrPA is a nonspecific inhibitor, affecting enzymes of different metabolic pathways in R. microplus. In our experiments, we demonstrated that 3-BrPA was able to affect the glycolytic enzyme hexokinase, reducing its activity by approximately 50%; and it strongly inhibited triose phosphate isomerase, which is an enzyme involved in both glycolysis and gluconeogenesis. Also, the mitochondrial respiratory chain was affected, NADH cytochrome c reductase (complex I-III) and succinate cytochrome c reductase (complex II-III) were strongly inhibited by 3-BrPA. Glutamate dehydrogenase was also affected by 3-BrPA, showing a gradual inhibition of activity in all the 3-BrPA concentrations tested. Altogether, these results show that 3-BrPA is a harmful compound to the tick organism.

Hepatotoxicity and nephrotoxicity of 3-bromopyruvate in mice

PURPOSE:To investigate the hepatotoxicity and nephrotoxicity of 3-Bromopyruvate (3BP) in mice.METHODS:Fifteen nude mice were grafted subcutaneously in the left flank with MDA-MB-231 cells, then all mice were divided into control group (PBS), 3BP group (8 mg/kg), positive group (DNR: 0.8 mg/kg) when tumor volume reached approximately 100 mm3. 28 days later, tumors, livers and kidneys were stored in 4 % formalin solution and stained with hematoxylin and eosin staining. The Kunming mice experiment included control group (PBS), 3BP group (4mg/kg; 8mg/kg; 16mg/kg), positive group (DNR: 0.8 mg/kg). 24 hours later, the blood were used for the determination of hepatic damage serum biomarkers. Livers were stored in 4 % formalin solution for the later detection.RESULTS:3BP at the dose of 8mg/kg had a good effect on inhibiting tumor growth in nude mice and did not damage liver and kidney tissues. Kunming mice experiment showed 3BP at the dose of 16mg/kg did damage to liver tissues.CONCLUSION:3-Bromopyruvate at the dose of suppressing tumor growth did not exhibit hepatotoxicity and nephrotoxicity in nude mice, and the effect on liver was confirmed in Kunming mice.(AU)

Hepatotoxicity and nephrotoxicity of 3-bromopyruvate in mice

ABSTRACT PURPOSE: To investigate the hepatotoxicity and nephrotoxicity of 3-Bromopyruvate (3BP) in mice. METHODS: Fifteen nude mice were grafted subcutaneously in the left flank with MDA-MB-231 cells, then all mice were divided into control group (PBS), 3BP group (8 mg/kg), positive group (DNR: 0.8 mg/kg) when tumor volume reached approximately 100 mm3. 28 days later, tumors, livers and kidneys were stored in 4 % formalin solution and stained with hematoxylin and eosin staining. The Kunming mice experiment included control group (PBS), 3BP group (4mg/kg; 8mg/kg; 16mg/kg), positive group (DNR: 0.8 mg/kg). 24 hours later, the blood were used for the determination of hepatic damage serum biomarkers. Livers were stored in 4 % formalin solution for the later detection. RESULTS: 3BP at the dose of 8mg/kg had a good effect on inhibiting tumor growth in nude mice and did not damage liver and kidney tissues. Kunming mice experiment showed 3BP at the dose of 16mg/kg did damage to liver tissues. CONCLUSION: 3-Bromopyruvate at the dose of suppressing tumor growth did not exhibit hepatotoxicity and nephrotoxicity in nude mice, and the effect on liver was confirmed in Kunming mice.

3-Bromopyruvic Acid Inhibits Tricarboxylic Acid Cycle and Glutaminolysis in HepG2 Cells.

BACKGROUND/AIM: 3-bromopyruvate (3BrPA) is an antitumor agent able to inhibit aerobic glycolysis and oxidative phosphorylation, therefore inducing cell death. However, cancer cells are also highly dependent of glutaminolysis and tricarboxylic acid cycle (TCA) regarding survival and 3BrPA action in these metabolic routes is poorly understood. MATERIALS AND METHODS: The effect of 3BrPA was characterized in mice liver and kidney mitochondria, as well as in human HepG2 cells. RESULTS: Low concentration of 3-BrPA significantly affected both glutaminolysis and TCA cycle functions, through inhibition of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and succinate dehydrogenase. Additionally, 3-BrPA treatment significantly decreased the reduced status of thiol groups in HepG2 cells without proportional increase of oxidizing groups, suggesting that these chemical groups are the target of alkylation reactions induced by 3-BrPA. CONCLUSION: This work demonstrates, for the first time, the effect of 3-BrPA in glutaminolysis and TCA cycle. Our results suggest that the combined action of 3-BrPA in glutaminolysis, TCA and glycolysis, inhibiting steps downstream of the glucose and glutamine metabolism, has an antitumor effect.