Shikonin alleviates choroidal neovascularization by inhibiting proangiogenic factor production from infiltrating macrophages.

Choroidal neovascularization (CNV), a feature of neovasular age-related macular degeneration (AMD), acts as a leading cause of vision loss in the elderly. Shikonin (SHI), a natural bioactive compound extracted from Chinese herb radix arnebiae, exerts anti-inflammatory and anti-angiogenic roles and also acts as a potential pyruvate kinase M2 (PKM2) inhibitor in macrophages. The major immune cells macrophages infiltrate the CNV lesions, where the production of pro-angiognic cytokines from macrophage facilitates the development of CNV. PKM2 contributes to the neovascular diseases. In this study, we found that SHI oral gavage alleviated the leakage, area and volume of mouse laser-induced CNV lesion and inhibited macrophage infiltration without ocular cytotoxicity. Moreover, SHI inhibited the secretion of pro-angiogenic cytokine, including basic fibroblast growth factor (FGF2), insulin-like growth factor-1 (IGF1), chemokine (C-C motif) ligand 2 (CCL2), placental growth factor and vascular endothelial growth factor (VEGF), from primary human macrophages by down-regulating PKM2/STAT3/CD163 pathway, indicating a novel potential therapy strategy for CNV.

Identification of the antibacterial mechanism of cryptotanshinone on methicillin-resistant Staphylococcus aureus using bioinformatics analysis.

Cryptotanshinone (CT) is an extract from the traditional Chinese medicine Salvia miltiorrhiza, which inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) in vitro. This study aims to determine the antibacterial mechanisms of CT by integrating bioinformatics analysis and microbiology assay. The microarray data of GSE13203 was retrieved from the Gene Expression Omnibus (GEO) database to screen the differentially expressed genes (DEGs) of S. aureus strains that were treated with CT treatment. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to identify the potential target of CT. Data mining on the microarray dataset indicated that pyruvate kinase (PK) might be involved in the antimicrobial activities of CT. The minimum inhibition concentrations (MICs) of CT or vancomycin against the MRSA strain ATCC43300 and seven other clinical strains were determined using the broth dilution method. The effects of CT on the activity of PK were further measured. In vitro tests verified that CT inhibited the growth of an MRSA reference strain and seven other clinical strains. CT hampered the activity of the PK of ATCC43300 and five clinical MRSA strains. CT might hinder bacterial energy metabolism by inhibiting the activity of PK.

Identification of Parthenolide Dimers as Activators of Pyruvate Kinase M2 in Xenografts of Glioblastoma Multiforme in Vivo.

Herein we detail the discovery of a series of parthenolide dimers as activators of PKM2 and evaluation of their anti-GBM activities. The most promising compound 5 showed high potency to activate PKM2 with an AC50 value of 15 nM, inhibited proliferation and metastasis, and induced apoptosis of GBM cells. Compound 5 could promote tetramer formation of PKM2 and reduce nucleus translocation of PKM2 in GBM cells without influence on the expression of total PKM2, thereby inhibiting the STAT3 signal pathway in vitro and in vivo. PKM2 knockdown assay demonstrated that the anti-GBM effect of 5 mainly depended on the expression of PKM2 in vitro and in vivo. Compound 16, a prodrug of 5, markedly suppressed U118 tumor xenograft growth and reduced the weight of tumor. On the basis of these investigations, we propose that 16 might be considered as a promising lead compound for discovery of anti-GBM drugs.

Virtual high-throughput screens identifying hPK-M2 inhibitors: Exploration of model extrapolation.

Glycolysis with PK-M2 occurs typically in anaerobic conditions and atypically in aerobic conditions, which is known as the Warburg effect. The Warburg effect is found in many oncogenic situations and is believed to provide energy and biomass for oncogenesis to persist. The work presented targets human PK-M2 (hPK-M2) in a virtual high-throughput screen to identify new inhibitors and leads for further study. In the initial screen, one of the 12 candidates selected for experimental validation showed biological activity (hit-rate = 8.13%). In the second screen with retrained models, six of 11 candidates selected for experimental validation showed biological activity (hit-rate: 54.5%). Additionally, four different scaffolds were identified for further analysis when examining the tested candidates and compounds in the training data. Finally, extrapolation was necessary to identify a sufficient number of candidates to test in the second screen. Examination of the results suggested stepwise extrapolation to maximize efficiency.

Novel antibacterial modalities against methicillin resistant Staphylococcus aureus derived from plants.

Methicillin-resistant Staphylococcus aureus (MRSA) is a notorious bacterial pathogen that induces high mortality and morbidity. Due to the emergence of multiple resistance, antibiotic treatments are rapidly becoming ineffective for the related infections. Natural products, especially those derived from plants, have been proven to be effective agents with unique antibacterial properties through different mechanisms. This review interprets the resistance mechanisms of MRSA with the aim to conquer public health threat. Further, recent researches about plant antimicrobials that showed remarkable antibacterial activity against MRSA are recorded, including the crude plant extracts and purified plant-derived bioactive compounds. Novel anti-MRSA modalities of plant antimicrobials such as alteration in efflux pump, inhibition of pyruvate kinase, and disturbance of quorum sensing in MRSA are also summarized which may be promising alternatives to antibacterial drug development in future.

Shikonin Inhibits Tumor Growth in Mice by Suppressing Pyruvate Kinase M2-mediated Aerobic Glycolysis.

Shift metabolism profile from mitochondrial oxidative phosphorylation to aerobic glycolysis (Warburg effect) is a key for tumor cell growth and metastasis. Therefore, suppressing the tumor aerobic glycolysis shows a great promise in anti-tumor therapy. In the present study, we study the role of shikonin, a naphthoquinone isolated from the traditional Chinese medicine Lithospermum, in inhibiting tumor aerobic glycolysis and thus tumor growth. We found that shikonin dose-dependently inhibited glucose uptake and lactate production in Lewis lung carcinoma (LLC) and B16 melanoma cells, confirming the inhibitory effect of shikonin on tumor aerobic glycolysis. Treatment of shikonin also decreased tumor cell ATP production. Furthermore, pyruvate kinase M2 (PKM2) inhibitor or activator respectively altered the effect of shikonin on tumor cell aerobic glycolysis, suggesting that suppression of cell aerobic glycolysis by shikonin is through decreasing PKM2 activity. Western blot analysis confirmed that shikonin treatment reduced tumor cell PKM2 phosphorylation though did not reduce total cellular PKM2 level. In vitro assay also showed that shikonin treatment significantly promoted tumor cell apoptosis compared to untreated control cells. Finally, when mice implanted with B16 cells were administered with shikonin or control vehicle, only shikonin treatment significantly decreased B16 tumor cell growth. In conclusion, this study demonstrates that shikonin inhibits tumor growth in mice by suppressing PKM2-mediated aerobic glycolysis.

2'-hydroxycinnamaldehyde inhibits cancer cell proliferation and tumor growth by targeting the pyruvate kinase M2.

It is reported that 2'-hydroxycinnamaldehyde (HCA), isolated from cinnamon, has anti-tumor effects through the modulation of multi-target molecules. In this study, we identified pyruvate kinase M2 (PKM2) as a direct target of HCA by use of biochemical methods including affinity chromatography, drug affinity responsive target stability, and cellular thermal shift assay. PKM2 is up-regulated in multiple cancer types and is considered as a potential target for cancer therapy. HCA binds directly to PKM2 and selectively decreases the phosphorylation of PKM2 at Tyr105, indicating a potential anti-proliferative effect on prostate cancer cells. As a PKM2 activator, HCA increases pyruvate kinase activity by promoting the tetrameric state of PKM2. However, HCA suppresses protein kinase activity of PKM2 by decreasing the phosphorylation at Tyr105. Moreover, this leads to a decrease of PKM2-mediated STAT3 phosphorylation at Tyr705 and a down-regulation of target genes, including MEK5 and cyclin D1. Furthermore, HCA suppresses tumor growth and the release of tumor extracellular vesicles in vivo by inhibiting the phosphorylation of PKM2. Collectively, our results suggest that HCA may be a potential anticancer agent targeting PKM2 in cancer progression.

Scutellarin inhibits Hela cell growth and glycolysis by inhibiting the activity of pyruvate kinase M2.

Scutellarin, one of natural flavonoids, is widely and clinically used for treating many diseases in China. Recently, scutellarin has demonstrated a broad spectrum of anti-proliferative activities against multiple cancer cell lines. However, the molecular mechanism of action remains to be investigated. We herein report the design and synthesis of biotinylated scutellareins as probes, which can be applied to discover scutellarein interacting proteins. Finally, we show that scutellarin directly targets pyruvate kinase M2 (PKM2) and inhibits its cytosolic activity to decrease glycolytic metabolism; on the other hand, scutellarin may also participate in regulating cell cycle and apoptotic proteins by activating MEK/ERK/PIN1 signaling pathway to promote the nuclear translocation of PKM2.

Differential kinetics at PK/PEPCK branch point in the cestode, Raillietina echinobothrida.

Pyruvate kinase (PK; EC 2.7.1.40) and phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are essential regulatory enzymes of glucose oxidation in helminths, the PK/PEPCK branch point being the first divergent step between carbohydrate catabolism of the parasites and their hosts. Recently, PEPCK from the cestode parasite, Raillietina echinobothrida, has been purified and characterized. In order to find out the differential kinetics, if any, at PK/PEPCK branch point in the parasite, in this study, we purified and characterized the parasite PK and compared it with the parasite PEPCK. The purified PK displayed standard Michaelis-Menten kinetics with Kmapp of 77.8 µM for its substrate PEP, whereas the Kmapp was 46.9 µM for PEPCK. PEP exhibited differential kinetics at PK/PEPCK branch point of the parasite and behaved as a homotropic effector for PEPCK, but not for PK. The inhibitory constant (Ki) for genistein and daidzein (phytochemicals from Flemingia vestita) was determined and discussed. From these results, we hypothesize that PK/PEPCK branch point is a probable site for anthelmintic action.

Expression, purification and characterization of Solanum tuberosum recombinant cytosolic pyruvate kinase.

The cDNA encoding for a Solanum tuberosum cytosolic pyruvate kinase 1 (PKc1) highly expressed in tuber tissue was cloned in the bacterial expression vector pProEX HTc. The construct carried a hexahistidine tag in N-terminal position to facilitate purification of the recombinant protein. Production of high levels of soluble recombinant PKc1 in Escherichia coli was only possible when using a co-expression strategy with the chaperones GroES-GroEL. Purification of the protein by Ni(2 +) chelation chromatography yielded a single protein with an apparent molecular mass of 58kDa and a specific activity of 34unitsmg(-1) protein. The recombinant enzyme had an optimum pH between 6 and 7. It was relatively heat stable as it retained 80% of its activity after 2min at 75°C. Hyperbolic saturation kinetics were observed with ADP and UDP whereas sigmoidal saturation was observed during analysis of phosphoenolpyruvate binding. Among possible effectors tested, aspartate and glutamate had no effect on enzyme activity, whereas α-ketoglutarate and citrate were the most potent inhibitors. When tested on phosphoenolpyruvate saturation kinetics, these latter compounds increased S0.5. These findings suggest that S. tuberosum PKc1 is subject to a strong control by respiratory metabolism exerted via citrate and other tricarboxylic acid cycle intermediates.

Discovery of a 1,2-bis(3-indolyl)ethane that selectively inhibits the pyruvate kinase of methicillin-resistant Staphylococcus aureus over human isoforms.

Methicillin-resistant Staphylococcus aureus pyruvate kinase (MRSA PK) has recently been identified as a target for development of novel antibacterial agents. Testing a series of 1,2-bis(3-indolyl)ethanes against MRSA PK has led to the discovery of a potent inhibitor that is selective over human isoforms.

Synergistic effects of diosmetin with erythromycin against ABC transporter over-expressed methicillin-resistant Staphylococcus aureus (MRSA) RN4220/pUL5054 and inhibition of MRSA pyruvate kinase.

Increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) worldwide with limited therapeutic options is a growing public health concern. Natural products have been shown to possess antibacterial actions against MRSA. Flavonoids from natural products have been shown to possess antibacterial actions against MRSA by antagonizing its resistance mechanisms. Diosmin and diosmetin are natural flavonoids found in a variety of citrus fruits. The aim of this study was to investigate whether diosmin and diosmetin could inhibit the growth of MRSA and the in vitro enzymatic activity of a newly discovered MRSA drug target, pyruvate kinase (PK). By using a panel of MRSA strains with known resistant mechanisms, neither diosmin nor diosmetin was shown to possess direct antibacterial activities against all tested MRSA strains. However, in checkerboard assay, we found that diosmetin together with erythromycin, could synergistically inhibit the growth of ABC-pump overexpressed MRSA-RN4220/pUL5054, and time kill assay also showed that the antibacterial activities of diosmetin with erythromycin were bactericidal. Diosmetin was further shown to significantly suppress the MRSA PK activities in a dose dependent manner. In conclusion, the inhibition of MRSA PK by diosmetin could lead to a deficiency of ATP and affect the bacterial efflux pump which might contribute to the antibacterial actions of diosmetin against MRSA.

Synergistic effects of baicalein with ciprofloxacin against NorA over-expressed methicillin-resistant Staphylococcus aureus (MRSA) and inhibition of MRSA pyruvate kinase.

ETHNOPHARMACOLOGICAL RELEVANCE: Baicalein, the active constituent derived from Scutellaria baicalensis Georgi., has previously been shown to significantly restore the effectiveness of ß-lactam antibiotics and tetracycline against methicillin-resistant Staphylococcus aureus (MRSA). With multiple therapeutic benefits, the antibacterial actions of baicalein may also be involved in overcoming other bacterial resistance mechanisms. The aim of the present study was to further investigate antibacterial activities of baicalein in association with various antibiotics against selected Staphylococcus aureus strains with known specific drug resistance mechanisms. MATERIAL AND METHODS: A panel of clinical MRSA strains was used for further confirmation of the antibacterial activities of baicalein. The effect of baicalein on inhibiting the enzymatic activity of a newly discovered MRSA-specific pyruvate kinase (PK), which is essential for Staphylococcus aureus growth and survival was also examined. RESULTS: In the checkerboard dilution test and time-kill assay, baicalein at 16 µg/ml could synergistically restore the antibacterial actions of ciprofloxacin against the NorA efflux pump overexpressed SA-1199B, but not with the poor NorA substrate, pefloxacin. Moreover, synergistic effects were observed when baicalein was combined with ciprofloxacin against 12 out of 20 clinical ciprofloxacin resistant strains. For MRSA PK studies, baicalein alone could inhibit the enzymatic activity of MRSA PK in a dose-dependent manner. CONCLUSION: Our results demonstrated that baicalein could significantly reverse the ciprofloxacin resistance of MRSA possibly by inhibiting the NorA efflux pump in vitro. The inhibition of MRSA PK by baicalein could lead to a deficiency of ATP which might further contribute to the antibacterial actions of baicalein against MRSA.

Protective effect of antioxidants on cerebrum oxidative damage caused by arginine on pyruvate kinase activity.

We have demonstrated that acute arginine administration decreases antioxidant defenses and compromises enzymes of respiratory chain in rat brain. In this study we evaluated in vivo and in vitro effect of arginine on pyruvate kinase activity, as well as its effect on an important parameter of oxidative stress namely thiobarbituric acid-reactive substances (TBA-RS) in cerebrum of rats. We also tested the influence of antioxidants, namely alpha -tocopherol plus ascorbic acid on the effects elicited by arginine in order to investigate the possible participation of free radicals on the effects of arginine on these parameters. Results showed that arginine acute administration inhibited pyruvate kinase activity in cerebrum of rats, as well as increased TBA-RS. By the other hand, arginine added to the incubation medium, in vitro studies, did not alter these parameters in rat cerebrum. In addition, pretreatment with antioxidants prevented the reduction of pyruvate kinase activity and the increase of TBA-RS caused by arginine. The data indicate that acute administration of arginine induces lipid peroxidation in rat cerebrum and that the inhibition of pyruvate kinase activity caused by this amino acid was probably mediated by free radicals since antioxidants prevented such effect. It is presumed that these results might be associated, at least in part, with the neuronal dysfunction of patients affected by hyperargininemia. Finally, we suggest that the administration of antioxidants should be considered as an adjuvant therapy to specific diets in hyperargininemia.

Characterization of the inhibition of pyruvate kinase caused by phenylalanine and phenylpyruvate in rat brain cortex.

Pyruvate kinase plays a crucial role on the glycolytic pathway, the main route that provides energy for brain functioning. In the present study, we investigated the kinetics of the inhibition of pyruvate kinase provoked by phenylalanine and its main metabolite, phenylpyruvate, in mitochondria-free cerebral cortex homogenate from 22-day-old Wistar rats. We found that phenylalanine and phenylpyruvate inhibit PK activity by competition with the enzyme substrates ADP and phosphoenolpyruvate. We also investigated the interaction between phenylalanine and phenylpyruvate, and the kinetics of alanine prevention of the inhibitory action of phenylalanine and phenylpyruvate on pyruvate kinase activity. We observed that alanine per se had no effect on PK activity but prevented the inhibitory action of phenylalanine and phenylpyruvate by competition. The data suggest that phenylalanine, phenylpyruvate, and alanine act on a common site in the enzyme, probably an allosteric one. It is possible that inhibition of brain PK activity may be related to the reduction of glucose metabolism observed in the brain of phenylketonuric patients and may be one of the mechanisms responsible for the neurological dysfunction found in these patients. Further studies, however, are necessary to evaluate the benefit of carbohydrate and alanine supplementation to the diet of phenylketonuric patients.

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes.

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).

Relationship between vitamin D status and deposition of bound calcium in skeletal muscle of the rat.

The effects of vitamin D on the intramuscular distribution of total and bound calcium, phosphate and on available cytosolic calcium, were investigated in skeletal muscle. Total calcium and phosphorus were measured on ashed subcellular fractions of muscles from vitamin D-repleted and vitamin D-deprived rats. The variations in available calcium were followed by determining the activities of calcium-sensitive enzymes in isolated cytosol. Bound-calcium was revealed ultra-microscopically by pyroantimonate. In vitamin D-repleted muscles, the pyroantimonate method revealed specific areas of intense bound-calcium deposition: the myofibrils, where they formed pronounced lines parallel to the Z-bands. In vitamin D-deficient muscles, the calcium-pyroantimonate deposits appeared clearly reduced. This loss was accompanied by a marked reduction in total calcium and phosphorus in all the subcellular fractions, as compared to vitamin D-repleted muscles. Unexpectedly, the activity of the Ca(+)-activated isocitrate-dehydrogenase was increased in the cytosol, while that of the Ca2(+)-inhibited pyruvate-kinase decreased. Prolonged vitamin D-administration to vitamin D-repleted rats led to an intensification of calcium-pyroantimonate deposits and a general increase in total calcium and phosphorus, but no change in the cytosolic Ca2(+)-sensitive enzyme activities. Cessation of vitamin D-administration to vitamin D-repleted rats produced a regression of calcium-pyroantimonate deposits, a general decrease of total calcium and phosphate levels, and stimulation of the Ca2(+)-activated isocitrate-dehydrogenase accompanied by lowering of the Ca2(+)-inhibited pyruvate-kinase. The results clearly indicate a correlation between vitamin D-repletion and the total and bound calcium content of skeletal muscle. In addition, they demonstrate an apparent contradiction between the decrease of total and bound calcium, and the activities of cytosolic Ca2+ sensitive enzymes during vitamin D-deprivation, which can only be explained by an increase in available calcium. It is suggested that vitamin D stimulates intramuscular mechanisms tending to lower available calcium by inactivating the cation via the formation of calcium chelates.