Aspirin modulates 2-hydroxyisobutyrylation of ENO1K281 to attenuate the glycolysis and proliferation of hepatoma cells.

Aspirin can efficiently inhibit the glycolysis and proliferation of cancer cells, however, the underlying mechanism is poorly understood. Here, we report that aspirin attenuates the glycolysis and proliferation of hepatoma cells through modulating the levels of lysine 2-hydroxyisobutyrylation (Khib) of enolase 1 (ENO1). We found that aspirin decreased the levels of glucose consumption and lactate production in hepatoma cells. Moreover, 4 mM aspirin reduced the activities of ENO1, a key enzyme of glycolysis, and decreased the levels of ENO1 Khib in the cells. Interestingly, we identified that 4 mM aspirin could decrease the levels of Khib on many proteins by using pan Khib antibody in the cells. Interestingly, the activities of ENO1 could be rescued by the transient overexpression of ENO1, but not by ENO1 mutant (K281R). Moreover, we identified that the C646, an inhibitor of p300 which is a writer of Khib, could reduce the levels of ENO1 Khib, resulting in the decrease of ENO1 activities. The treatment with PDTC, an inhibitor of NF-κB which is a target of aspirin, could work well as C646 in the cells. Both of aspirin and C646 (or PDTC) displayed a stronger effect than the single treatment in the system. Functionally, ENO1, but not ENO1 mutant (K281R), could rescue the aspirin-induced inhibition of proliferation of liver cancer cells in vitro, suggesting that ENO1K281 is involved in the aspirin-mediated inhibition of liver cancer. Our finding provides new insights into the mechanism by which aspirin attenuates the glycolysis and proliferation of hepatoma cells.

Specific activation of glycolytic enzyme enolase 2 in BRAF V600E-mutated colorectal cancer.

The BRAF V600E mutation occurs in approximately 10% of patients with metastatic colorectal cancer (CRC) and constitutes a distinct subtype of the disease with extremely poor prognosis. To address this refractory disease, we investigated the unique metabolic gene profile of BRAF V600E-mutated tumors via in silico analysis using a large-scale clinical database. We found that BRAF V600E-mutated tumors exhibited a specific metabolic gene expression signature, including some genes that are associated with poor prognosis in CRC. We discovered that BRAF V600E-mutated tumors expressed high levels of glycolytic enzyme enolase 2 (ENO2), which is mainly expressed in neuronal tissues under physiological conditions. In vitro experiments using CRC cells demonstrated that BRAF V600E-mutated cells exhibited enhanced dependency on ENO2 compared to BRAF wild-type cancer cells and that knockdown of ENO2 led to the inhibition of proliferation and migration of BRAF V600E-mutated cancer cells. Moreover, inhibition of ENO2 resulted in enhanced sensitivity to vemurafenib, a selective inhibitor of BRAF V600E. We identified AP-1 transcription factor subunit (FOSL1) as being involved in the transcription of ENO2 in CRC cells. In addition, both MAPK and PI3K/Akt signaling were suppressed upon inhibition of ENO2, implying an additional oncogenic role of ENO2. These results suggest the crucial role of ENO2 in the progression of BRAF V600E-mutated CRC and indicate the therapeutic implications of targeting this gene.

In vitro inhibition studies of coumarin derivatives on Bos taurus enolase and elucidating their interaction by molecular docking, molecular dynamics simulations and MMGB(PB)SA binding energy calculation.

Tropical theileriosis is among the most common vector-borne diseases and caused by Theileria parasites. Theileria annulata is an obligate intracellular protozoan parasite and transmitted to especially Bos taurus and Bos indicus by Hyalomma tick vectors. C8 ([4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one); C9 (4-(3,4-dihydroxyphenyl)-7,8 dihydroxy-2H-chromen-2-one); C21 (4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2 one) were identified as potent Theileria annulata enolase (TaEno) inhibitors in our previous studies. An ideal drug compound must inhibit the target parasite enzyme without inhibiting its homolog in the host. In this study, the inhibitory effect of the compounds previously evaluated on TaEno were tested on the host Bos taurus enolase (BtEno3) by in vitro studies. The interactions of enzyme-coumarin and enzyme-coumarin-substrate by in silico studies were also performed. All of the coumarin derivatives tested showed very low inhibitory effects on B. taurus enolase; 36,87% inhibition at 100 µM concentration for C8, 8,13% inhibition at 100 µM concentration for C9 and 77,69 µM of IC50 value for C21. In addition, these three coumarin derivatives and substrate 2PG were docked into the BtEno3 using molecular docking methods. Molecular interactions between enolase-coumarin and enolase-coumarin-substrate complexes were analyzed using molecular dynamics simulation methods for 100 ns. Estimated free energy of bindings of the substrate 2PG and coumarin derivatives to the BtEno3 were calculated by MM-GB(PB)SA methods. In comparison to the inhibition studies performed on TaEno, C8 and C9 coumarin derivatives remain the possible inhibitor candidates as they inhibit the host enolase at very high concentrations. These two promising compounds will be further analyzed by in vitro and in vivo studies towards developing an alternative drug against tropical theileriosis.

Molecular docking of alpha-enolase to elucidate the promising candidates against Streptococcus pneumoniae infection.

PURPOSE: To predict potential inhibitors of alpha-enolase to reduce plasminogen binding of Streptococcus pneumoniae (S. pneumoniae) that may lead as an orally active drug. S. pneumoniae remains dominant in causing invasive diseases. Fibrinolytic pathway is a critical factor of S. pneumoniae to invade and progression of disease in the host body. Besides the low mass on the cell surface, alpha-enolase possesses significant plasminogen binding among all exposed proteins. METHODS: In-silico based drug designing approach was implemented for evaluating potential inhibitors against alpha-enolase based on their binding affinities, energy score and pharmacokinetics. Lipinski's rule of five (LRo5) and Egan's (Brain Or IntestinaL EstimateD) BOILED-Egg methods were executed to predict the best ligand for biological systems. RESULTS: Molecular docking analysis revealed, Sodium (1,5-dihydroxy-2-oxopyrrolidin-3-yl)-hydroxy-dioxidophosphanium (SF-2312) as a promising inhibitor that fabricates finest attractive charges and conventional hydrogen bonds with S. pneumoniae alpha-enolase. Moreover, the pharmacokinetics of SF-2312 predict it as a therapeutic inhibitor for clinical trials. Like SF-2312, phosphono-acetohydroxamate (PhAH) also constructed adequate interactions at the active site of alpha-enolase, but it predicted less favourable than SF-2312 based on binding affinity. CONCLUSION: Briefly, SF-2312 and PhAH ligands could inhibit the role of alpha-enolase to restrain plasminogen binding, invasion and progression of S. pneumoniae. As per our investigation and analysis, SF-2312 is the most potent naturally existing inhibitor of S. pneumoniae alpha-enolase in current time.

An enolase inhibitor for the targeted treatment of ENO1-deleted cancers.

Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1-deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1-deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings.

Enolase inhibition alters metabolic hormones and inflammatory factors to promote neuroprotection in spinal cord injury.

Enolase inhibition is a potential therapeutic strategy currently being investigated for treatment of spinal cord injury (SCI) as it reduces pro-inflammatory cytokines and chemokines, alters metabolic factors, and reduces gliosis in acute SCI. Herein, the role of enolase in SCI has been examined to better understand the effects of this enzyme on inflammation, metabolic hormones, glial cell activation, and neuroprotection under these shorter injury conditions. Immunohistochemical analyses of inflammatory markers vimentin, Cox-2, and caspase-1 indicated that enolase inhibition attenuated the elevated levels of inflammation seen following SCI. Iba1, GFAP, NFP, and CSPG staining indicated that enolase inhibition with prolonged administration of ENOblock reduced microglia/astrocyte activation and lead to enhanced neuroprotection in SCI. An analysis of metabolic hormones revealed that ENOblock treatment significantly upregulated plasma concentrations of peptide YY, glucagon-like peptide 1, glucose-dependent insulinotropic peptide, glucagon, and insulin hormones as compared to vehicle-treated controls (Mann-Whitney, p ≤ 0.05). ENOblock did not have a significant effect on plasma concentrations of pancreatic polypeptide. Interestingly, ENOblock treatment inhibited chondroitin sulfate proteoglycan (CSPG), which is produced by activated glia and serves to block regrowth of axons across the lesion site following injury. An increased level of NeuN and MBP with reduced caspase-1 was detected in SCI tissues after ENOblock treatment, suggesting preservation of myelin and induction of neuroprotection. ENOblock also induced improved motor function in SCI rats, indicating a role for enolase in modulating inflammatory and metabolic factors in SCI with important implications for clinical consideration.

Generation and Characterization of Single Chain Variable Fragment against Alpha-Enolase of Candida albicans.

Candida albicans (C. albicans) is an opportunistic human pathogen responsible for approximately a half of clinical candidemia. The emerging Candida spp. with resistance to azoles is a major challenge in clinic, suggesting an urgent demand for new drugs and therapeutic strategies. Alpha-enolase (Eno1) is a multifunctional protein and represents an important marker for invasive candidiasis. Thus, C. albicans Eno1 (CaEno1) is believed to be an important target for the development of therapeutic agents and antibody drugs. Recombinant CaEno1 (rCaEno1) was first used to immunize chickens. Subsequently, we used phage display technology to construct two single chain variable fragment (scFv) antibody libraries. A novel biopanning procedure was carried out to screen anti-rCaEno1 scFv antibodies, whose specificities were further characterized. The polyclonal IgY antibodies showed binding to rCaEno1 and native CaEno1. A dominant scFv (CaS1) and its properties were further characterized. CaS1 attenuated the growth of C. albicans and inhibited the binding of CaEno1 to plasminogen. Animal studies showed that CaS1 prolonged the survival rate of mice and zebrafish with candidiasis. The fungal burden in kidney and spleen, as well as level of inflammatory cytokines were significantly reduced in CaS1-treated mice. These results suggest CaS1 has potential of being immunotherapeutic drug against C. albicans infections.

Preclinical validation of Alpha-Enolase (ENO1) as a novel immunometabolic target in multiple myeloma.

Bone marrow plasmacytoid dendritic cells (pDCs) in patients with multiple myeloma (MM) promote tumor growth, survival, drug resistance, and immune suppression. Understanding the molecular signaling crosstalk among the tumor cells, pDCs and immune cells will identify novel therapeutic approaches to enhance anti-MM immunity. Using oligonucleotide arrays, we found that pDC-MM interactions induce metabolic enzyme Alpha-Enolase (ENO1) in both pDCs and MM cells. Analysis of MM patient gene expression profiling database showed that ENO1 expression inversely correlates with overall survival. Protein expression analysis showed that ENO1 is expressed in pDC and MM cells; and importantly, that pDC-MM coculture further increases ENO1 expression in both MM cells and pDCs. Using our coculture models of patient autologous pDC-T-NK-MM cells, we examined whether targeting ENO1 can enhance anti-MM immunity. Biochemical inhibition of ENO1 with ENO1 inhibitor (ENO1i) activates pDCs, as well as increases pDC-induced MM-specific CD8+ CTL and NK cell activity against autologous tumor cells. Combination of ENO1i and anti-PD-L1 Ab or HDAC6i ACY-241 enhances autologous MM-specific CD8+ CTL activity. Our preclinical data therefore provide the basis for novel immune-based therapeutic approaches targeting ENO1, alone or in combination with anti-PD-L1 Ab or ACY241, to restore anti-MM immunity, enhance MM cytotoxicity, and improve patient outcome.

In silico prediction of a new lead compound targeting enolase of trypanosomatids through structure-based virtual screening and molecular dynamic studies.

Enolase is one of the key glycolytic metalloenzyme in many organisms, and it is a potential therapeutic target including trypanosomatids. Sequence and structural analysis of enolase of Trypanosoma bruzi (TbENO), Trypanosoma cruzi (TcENO) and Leishmania donovani (LdENO) revealed conserved sequence pattern and structural features. Hence identification of an inhibitor against enolase of one trypanosomatid organism may have similar effects on enolase of homologous organisms belonging to same family. In the process to identify potent inhibitor compounds against TbENO by in silico methods, compounds containing the substructures of substrate, i.e. phosphoenolpyruvate (PEP) and the well-known inhibitors, fluoro-2-phosphono-acetohydroxamate (FPAH) and phosphono-acetohydroxamate (PAH), were collected. Virtual screening and induced fit docking studies were carried out to explore compounds that have better binding affinity than PEP and FPAH. PPPi was found to be the top hit exhibiting significant binding affinity towards enolase. Glide energy values of two other compounds represented by PubChem ID: 511392 and 101803456 was in good agreement with PEP and PAH. TbENO-PPPi complex was subjected to molecular orbital analysis and molecular dynamic studies by considering its remarkable binding affinity as it could be a potent inhibitor of enolase. Despite being an endogenous compound, based on the results of this study, we highlight PPPi to be a lead compound, and its structure can be treated as a model for further chemical modifications to obtain more potent antagonists.

Discovery and evaluation of inhibitory activity and mechanism of arylcoumarin derivatives on Theileria annulata enolase by in vitro and molecular docking studies.

In this study, the inhibition potential of 3- and 4-arylcoumarin derivatives on Theileria annulata enolase (TaENO) was assessed for the first time in the literature. Firstly, protein stabilization analyses of TaENO were performed and it was found that the enzyme remains stable with the addition of 6 M ethylene glycol at + 4 °C. Inhibitor screening analyses were carried out using 25 coumarin derivatives on highly purified TaENO (> 95%), and four coumarin derivatives [4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one (C8); 4-(3,4-dihydroxyphenyl)-7,8 dihydroxy-2H-chromen-2-one (C9); 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2 one (C21); and 3-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one (C23)] showed the highest inhibitory effects with the IC50 values of 10.450, 13.170, 8.871 and 10.863 µM, respectively. The kinetic results indicated that these compounds inhibited the enzyme by uncompetitive inhibition. In addition, the successful binding of the most potent inhibitor (C21) into TaENO was confirmed by using MALDI-TOF mass spectrophotometry. Molecular docking analyses have predicted that C8 and C21 coumarin derivatives which showed high inhibitory effects on TaENO were interacted with high affinity to the potential regions out of the active site. Taken together, these coumarin derivatives (C8, C9, C21 and C23) are first known potent, nonsubstrate, uncompetitive inhibitors of TaENO and these results will facilitate further in vitro and in vivo analysis toward structure-based drug design studies.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

The 3S Enantiomer Drives Enolase Inhibitory Activity in SF2312 and Its Analogues.

We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.

Structural and Functional Studies of Bacterial Enolase, a Potential Target against Gram-Negative Pathogens.

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, ß-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and ß-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 µg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring ß-thujaplicin and the α- and ß-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 µM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.

ENOblock inhibits the pathology of diet-induced obesity.

Obesity is a medical condition that impacts on all levels of society and causes numerous comorbidities, such as diabetes, cardiovascular disease, and cancer. We assessed the suitability of targeting enolase, a glycolysis pathway enzyme with multiple, secondary functions in cells, to treat obesity. Treating adipocytes with ENOblock, a novel modulator of these secondary 'moonlighting' functions of enolase, suppressed the adipogenic program and induced mitochondrial uncoupling. Obese animals treated with ENOblock showed a reduction in body weight and increased core body temperature. Metabolic and inflammatory parameters were improved in the liver, adipose tissue and hippocampus. The mechanism of ENOblock was identified as transcriptional repression of master regulators of lipid homeostasis (Srebp-1a and Srebp-1c), gluconeogenesis (Pck-1) and inflammation (Tnf-α and Il-6). ENOblock treatment also reduced body weight gain, lowered cumulative food intake and increased fecal lipid content in mice fed a high fat diet. Our results support the further drug development of ENOblock as a therapeutic for obesity and suggest enolase as a new target for this disorder.

Alpha-enolase regulates the malignant phenotype of pulmonary artery smooth muscle cells via the AMPK-Akt pathway.

The molecular mechanisms underlying the metabolic shift toward increased glycolysis observed in pulmonary artery smooth muscle cells (PASMC) during the pathogenesis of pulmonary arterial hypertension (PAH) are not fully understood. Here we show that the glycolytic enzyme α-enolase (ENO1) regulates the metabolic reprogramming and malignant phenotype of PASMC. We show that ENO1 levels are elevated in patients with associated PAH and in animal models of hypoxic pulmonary hypertension (HPH). The silencing or inhibition of ENO1 decreases PASMC proliferation and de-differentiation, and induces PASMC apoptosis, whereas the overexpression of ENO1 promotes a synthetic, de- differentiated, and apoptotic-resistant phenotype via the AMPK-Akt pathway. The suppression of ENO1 prevents the hypoxia-induced metabolic shift from mitochondrial respiration to glycolysis in PASMC. Finally, we find that pharmacological inhibition of ENO1 reverses HPH in mice and rats, suggesting ENO1 as a regulator of pathogenic metabolic reprogramming in HPH.

Silencing of ENO1 by shRNA Inhibits the Proliferation of Gastric Cancer Cells.

α-Enolase is a significant subunit of enolase and acts as a glycolytic enzyme responsible for catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the anaerobic glycolysis pathway. The research about their role is known little in tumor invasion and metastasis. This research analyzed the effect of α-enolase in proliferation and progression of human gastric cancer. The constructed PLKO.1-ENO1 shRNA vector was transfected into 293 T cells and used to infect gastric cancer cells, MKN45, by using lentivirus method. Negative controls were generated by infection with viruses containing empty vector PLKO.1-scramble-shRNA by the same protocol and using wild-type MKN45 cells as blank control. The silencing effect was confirmed by reverse transcription polymerase chain reaction and Western blotting at messenger RNA and protein levels, respectively. Cell proliferation and chemosensitivity were tested by methyl-thiazolyl-tetrazolium assay. Cell apoptosis was tested by flow cytometry. The cell line α-enolase short hairpin RNA stabling silence α-enolase was successfully constructed. In the α-enolase short hairpin RNA cell lines, messenger RNA and protein expression of α-enolase were significantly lower than those in negative control and blank control groups. The proliferation and clone formation ability were significantly inhibited, cell apoptosis was increased significantly, and the inhibition rate of chemotherapy drugs was increased ( P < .05). Our data provide strong evidence that α-enolase short hairpin RNA interference vector can effectively suppress the proliferation and increase chemosensitivity of MKN45 cells, which may provide a novel gene therapy for gastric cancer.

ENO2 Promotes Cell Proliferation, Glycolysis, and Glucocorticoid-Resistance in Acute Lymphoblastic Leukemia.

BACKGROUND/AIMS: The metabolic features of cancer cells have long been acknowledged to be altered and to provide new therapeutic opportunities. The expression of glycolytic enzyme enolase 2 (ENO2) was found to be closely associated with the clinical features of acute lymphoblastic leukemia (ALL) patients, but its functions remain unclear in ALL. METHODS: We evaluated the association between ENO2 mRNA expression in bone marrow mononuclear cells (BM-MNCs) and the efficacy of chemotherapy, and further explored the function of ENO2 in ALL. The molecular mechanisms of ENO2 expression and its effects on cell growth, glycolysis and glucocorticoid resistance were explored by Cell Counting Kit-8, glucose-consumption assay, Quantitative RT-PCR, Western blotting and in vivo tumorigenesis in NOD/SCID mice. RESULTS: The results showed that ENO2 mRNA expression in BM-MNCs was significantly decreased when patients completed induction chemotherapy and reached complete remission (CR). ENO2 mRNA expression was increased when patients suffered relapse. Functional studies demonstrated that ENO2 promoted cell growth, glycolysis, and glucocorticoid resistance, all of which were effectively inhibited when ENO2 was silenced with shRNAs. Further studies revealed that ENO2 up-regulated various glycolysis-related genes and enhanced Akt activity with subsequent glycogen synthase kinase3ß (GSK-3ß) phosphorylation, inducing cell proliferation and glycolysis. The combination of silencing ENO2 and 2-deoxyglucose (2-DG) synergistically inhibited leukemia cell survival. CONCLUSIONS: These results indicate that ENO2 may be a biological marker for monitoring chemotherapeutic efficacy and relapse in ALL. ENO2 may provide a potential therapeutic strategy for ALL.

Enolase from Trypanosoma cruzi is inhibited by its interaction with metallocarboxypeptidase-1 and a putative acireductone dioxygenase.

Purification of enolase (ENO) from the cytosol of Trypanosoma cruzi indicated that it may interact with at least five other proteins. Two of them were identified as metallocarboxypeptidase-1 (TcMCP-1) and a putative acireductone dioxygenase (ARDp). Subcellular localization studies confirmed the presence of ARDp in the cytosol, as is the case for ENO and TcMCP-1. Analysis of the ARDp sequence showed that this protein has two domains, an N-terminal ARD and a C-terminal TRP14 (thioredoxin-related protein) domain. The interactions between ENO, TcMCP-1 and ARDp were confirmed for the natural proteins from the trypanosome (using size-exclusion chromatography and co-immunoprecipitation from a cytosolic fraction) and recombinant forms (using ELISA ligand-binding assay and ENO activity assays). The ELISA ligand-binding assays permitted to verify the optimal physicochemical conditions for the interactions (representative for the physiological conditions) and to determine the affinity constants (Kd): ENO/ARDp: 9.54 ±â€¯0.82 nM, ARDp/ENO 10.05 ±â€¯1.11 nM, and ENO/TcMCP-1: 5.66 ±â€¯0.61 nM. The data also show that the interaction between TcMCP-1 and ARDp is mediated by ENO acting as a "bridge". Furthermore, considerable inhibition of the ENO activity, up to 85%, is observed when the enzyme interacts with TcMCP-1 and ARDp simultaneously. All these data confirm that the interaction between ENO, TcMCP-1 and ARDp, occurring in T. cruzi's cytosol, modulates the ENO activity and suggest a possible physiological mechanism for regulation of the ENO activity by the protein-protein interaction.

Long-term stability of glucose: glycolysis inhibitor vs. gel barrier tubes.

BACKGROUND: Measuring the glucose concentration in whole blood samples is critical due to unsatisfactory glycolysis inhibition. Previous studies showed that Terumo tubes were superior, but they were taken off the European market in 2016 and alternatives were required. This initiated the present evaluation of glucose stability in five available tube types. METHODS: Venous blood samples were collected from 61 healthy volunteers to test tubes supplied by Terumo (two sets), Greiner FC-Mix, BD FX-Mixture and BD serum. After sampling, the contents were thoroughly mixed and centrifuged within an hour. The glucose concentrations were determined and the samples resuspended except for BD serum tubes (gel barrier). The first 30 samples were stored at room temperature and the remaining 31 at 4°C. After 24, 48, 72 and 96 h, all tubes were (re)centrifuged, and glucose concentration measurements were repeated. RESULTS: Changes in glucose concentrations over time differed significantly between the investigated tube types and to a certain extent between the two storing conditions. Glycolysis was most evident in the BD FX-mixture tubes. Good glucose stability was observed in samples retrieved form BD serum and Greiner tubes. The stability in both Terumo tubes was comparable to that in other studies. Although Greiner and both Terumo tubes are supposed to contain the same glycolysis inhibitor, glucose stability differed between these tubes. CONCLUSIONS: We showed that Greiner is an acceptable alternative to Terumo and that glucose in serum that was rapidly separated from corpuscles by a gel barrier is stable for an extended time.

In silico-based identification of human α-enolase inhibitors to block cancer cell growth metabolically.

Unlimited growth of cancer cells requires an extensive nutrient supply. To meet this demand, cancer cells drastically upregulate glucose uptake and metabolism compared to normal cells. This difference has made the blocking of glycolysis a fascinating strategy to treat this malignant disease. α-enolase is not only one of the most upregulated glycolytic enzymes in cancer cells, but also associates with many cellular processes or conditions important to cancer cell survival, such as cell migration, invasion, and hypoxia. Targeting α-enolase could simultaneously disturb cancer cells in multiple ways and, therefore, is a good target for anticancer drug development. In the current study, more than 22 million chemical structures meeting the criteria of Lipinski's rule of five from the ZINC database were docked to α-enolase by virtual screening. Twenty-four chemical structures with docking scores better than that of the enolase substrate, 2-phosphoglycerate, were further screened by the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties prediction. Four of them were classified as non-mutagenic, non-carcinogenic, and capable of oral administration where they showed steady interactions to α-enolase that were comparable, even superior, to the currently available inhibitors in molecular dynamics (MD) simulation. These compounds may be considered promising leads for further development of the α-enolase inhibitors and could help fight cancer metabolically.