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.

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.

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.

Electroacupuncture reduces hemiplegia following acute middle cerebral artery infarction with alteration of serum NSE, S-100B and endothelin.

Acupuncture may help motor recovery in chronic stroke survivors, but it is unclear whether it is useful for acute or subacute stroke patients. This study aimed to assess the effiency of electroacupuncture on hemiplegic patients caused by acute first-ever middle cerebral artery infarction. Ninety-eight patients with hemiplegia after first-ever middle cerebral artery infarction were divided into the observation group and the control group. Electroacupuncture was applied once daily for three weeks seven days after symptom onset. The motor functions of the limbs and the activities of daily living (ADL) were evaluated by Fugl-Meyer assessment (FMA) and Barthel index (BI). Serum neuron-specific enolase (NSE), soluble protein-100B (S-100B) and endothelin (ET) were quantified before and after treatment.After treatment, the FMA and BI scores were improved in comparison to before treatment scores in the same group (P<0.01 or P<0.05), with a more significant improvement in the observation group (with electroacupuncture) than in the control group (P<0.01). After treatments, the amounts of serum NSE, S-100B and ET in the observation group were significantly decreased when compared with those of the control group (P<0.01 or P<0.05). No adverse reactions occurred during electroacupuncture. This study showed that motor functions of the limbs and the activities of daily living in hemiplegic patients caused by acute cerebral infarction were improved significantly after treatment with electroacupuncture and this improvement was associated with reduced serum levels of NSE, S-100B and ET.

Utilization of substrate-induced quenching for screening targets promoting NADH and NADPH consumption.

Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappears with oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based on monitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.

UVA1-induced decrease in dermal neuron-specific enolase (NSE) in acrosclerosis.

Besides its role in small-cell carcinoma of the lung, elevated serum levels of neuron-specific enolase (NSE) have recently been reported to be associated with autoimmune rheumatic disorders such as systemic sclerosis. Serum NSE seems to correlate with disease activity as well as Rodnan skin score. The aim of the study was to assess the neuromodulatory effects of conventional UVA1 phototherapy on acrosclerosis as an additional mechanism besides an assumed T cell apoptosis, collagenase induction and angiogenesis. Punch skin biopsies of acrosclerotic skin lesions taken before and after treatment from four patients were evaluated immunohistochemically for the presence of NSE, S100 and neurofilament. Immunolabeling revealed a UVA-induced decrease in dermal NSE expression. In contrast, no alteration in neurofilament+ cells could be detected. In line with the findings of a previous investigation, a high number of S100+ cells were detected in most specimens. We demonstrated a UVA1-induced reduction in dermal NSE levels correlating with a softening of former sclerotic lesions. Even though the origin and the functional mechanisms remain obscure, NSE might be relevant directly within sclerotic skin lesions and may possibly be used as a diagnostic marker at least in SSc-associated acrosclerotic skin.

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).

Interaction of the ligand-activated glucocorticoid receptor with the 14-3-3 eta protein.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor. In this study, we used the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with the human GR ligand-binding domain (LBD) in a ligand-dependent manner. One isolated cDNA from a HeLa cell library encoded the COOH-terminal portion of the eta-isoform of the 14-3-3 protein (residues 187-246). Glucocorticoid agonists, triamcinolone acetonide and dexamethasone, induced the GR LBD/14-3-3eta protein fragment interaction, but an antagonist, RU486, did not. Glutathione S-transferase pull-down experiments in vitro showed that full-length 14-3-3eta protein also interacted with the activated GR. Transient transfection studies using COS-7 cells revealed a stimulatory effect of 14-3-3eta protein on transcriptional activation by the GR. The 14-3-3 family members have recently been found to associate with a number of important signaling proteins, such as protein kinase C and Raf-1, as functional modulators. Our findings suggest a novel regulatory role of 14-3-3eta protein in GR-mediated signaling pathways and also point to a mechanism whereby GR may cross-talk with other signal transduction systems.

Fluoride inhibition of yeast enolase: crystal structure of the enolase-Mg(2+)-F(-)-Pi complex at 2.6 A resolution.

Enolase in the presence of its physiological cofactor Mg2+ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase-Mg(2+)-F(-)-Pi has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/sigma (F) > or = 3 to 2.6 A resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-169, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate-enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg2+ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg2+. The synergism of inhibition by F- and Pi most probably is due to the requirement of Pi for the closed conformation. It is possible that other Mg(2+)-dependent enzymes that have OH- ions bound to the metal ion in the transition state also will be inhibited by fluoride ions.