Docking Simulation and Competitive Experiments Validate the Interaction Between the 2,5-Xylidine Inhibitor and Rigidoporus lignosus Laccase

Abstract

Laccases are polyphenol oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds, and their use in biotechnological applications is increasing.

Recently, the first X-ray structure of active laccase from white rot fungus Rigidoporus lignosus has been reported containing a full complement of copper ions. Comparison among selected fungal laccases of known 3D structure has shown that the Rigidoporus lignosus laccase has a very high similarity with the Trametes versicolor laccase that, being co-crystallized with 2,5-xylidine, shows a well defined binding pocket for the substrate. Global sequence alignment between Rigidoporus lignosus and Trametes versicolor laccases shows 73% of identity but, surprisingly, there is no identity and neither conservative substitutions between the residues composing the loops directly contacting the 2,5-xylidine. Moreover the structural alignment of these two enzymes identifies in these loops a striking structural similarity proposing the question if 2,5-xylidine may bind in same enzyme pocket.

Here we report the protein-ligand docking simulation of 3D structure of Rigidoporus lignosus laccase and 2,5-xylidine. Docking simulation analyses show that spatial conformation of the two 2,5-xylidine binding pockets, despite differences in the residues directly contacting the ligand, may arrange a similar pocket that allows a comparable accommodation of the inhibitor. To validate these results the binding of 2,5-xylidine in the substrate cavity has been confirmed by kinetic competitive experiments.     

Novel, lipophilic derivatives of 2,5-dideoxy-2,5-imino-D-mannitol (DMDP) are powerful beta-glucosidase inhibitors

Novel derivatives of the D-glucosidase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol bearing lipophilic aliphatic or aromatic amides attached to C-1 have been found to inhibit beta-glucosidase from Agrobacterium sp. in the nanomolar range. One of them, a coumarin derivative, ranks amongst the most active compounds in the class of reversible glycosidase inhibitors of the iminoalditol type.     

2,5-Anhydro-D-mannitol increases hepatocyte calcium: implications for a hepatic hunger stimulus

The fructose analogue, 2,5-anhydro-D-mannitol (2,5-AM), triggers feeding in rats via a mechanism linked to its ability to trap phosphate and deplete hepatic ATP. This metabolic inhibitor is particularly useful in the study of the role of the liver in initiation of feeding as its effects are preferentially localized to the liver, and its metabolic consequences have been extensively characterized. To determine whether changes in intracellular calcium may participate in a mechanism conveying information about hepatic energy status to the nervous system, we studied the effects of 2,5-AM on intracellular calcium in isolated hepatocytes using the ratiometric indicator, fura-2. 2,5-AM elicited a marked elevation of intracellular calcium within 2-3 min of exposure that returned to baseline upon removal of the agent. Removal of external calcium failed to prevent this response, while emptying intracellular stores prevented it. These data are consistent with the hypothesis that hepatic energy status may be conveyed to the nervous system via a calcium-mediated secretion event.     

In vitro binding of [14C]2,5-hexanedione to rat neuronal cytoskeletal proteins

2,5-Hexanedione (2,5-HD) induces central-peripheral axonpathy characterized by the accumulation of 10-nm neurofilaments proximal to the nodes of Ranvier and a Wallerian-type degeneration. It has been postulated that neurofilament crosslinking may be involved in the production of this axonopathy. A potential initiating event in this neurotoxic process may be the direct binding of 2,5-HD to neurofilament and microtubule proteins. In this study, the in vitro binding of [¹⁴C]2,5-HD to neurofilament and microtubule proteins was examined. Neurofilament proteins isolated from rat spinal cord or microtubule proteins isolated from rat brain were incubated in the presence of 2,5-HD at concentrations ranging 25 to 500 mM. Quantitative analysis of sodium dodecyl sulfate (SDS) polyacrylamide gels revealed a dose- and time-dependent binding of 2,5-HD to both neurofilament proteins and microtubule proteins. Expressed as pmol 2,5-HD bound per g protein, the observed relative binding was MAP2>NF160>NF200>NF68>tubulin. These data demonstrate the direct binding of 2,5-HD to cytoskeletal proteins including both neurofilaments and microtubules.     

Synthesis, biological evaluation, and molecular docking studies of 2,5-substituted-1,4-benzoquinone as novel urease inhibitors

A series of 2,5-substituted-1,4-benzoquinone (1-6) were prepared and structurally characterized by elemental analysis, IR spectra, (1)H and (13)C NMR spectra, and single crystal X-ray determination. The urease inhibitory activities of the compounds against H. pylori urease were studied. Among the compounds, 2,5-bis(2-morpholin-4-ylethylamino)-[1,4]benzoquinone (2) shows the most effective activity with IC(50) value of 27.30±2.17μM. Docking simulation was performed to insert compound 2 into the crystal structure of H. pylori urease at the active site to determine the probable binding mode. As a result, compound 2 may be used as a potential urease inhibitor.     

Inhibition of glycogenolysis by 2,5-anhydro-D-mannitol in isolated rat hepatocytes

2,5-Anhydro-D-mannitol, an analog of D-fructofuranose, inhibited basal and glucagon-stimulated glycogenolysis and glucose production in hepatocytes isolated from fed rats. Glucose formation from galactose was unaffected by the inhibitor. 2,5-Anhydro-D-mannitol-1-phosphate inhibits phosphorylase alpha with a Ki value of 2.4 mM. This same phosphorylated metabolite accumulates to the extent of 9.2 mumol/g wet wt in treated hepatocytes suggesting that phosphorolysis is the locus of the inhibition of glucose production from glycogen. Our results suggest that 2,5-anhydro-D-mannitol can be used to produce a model of hereditary fructose intolerance and that it merits further study as a hypoglycemic agent.     

Identification of 6-substituted 4-arylsulfonyl-1,4-diazepane-2,5-diones as a novel scaffold for human chymase inhibitors

A novel series of 6-substituted 4-sulfonyl-1,4-diazepane-2,5-diones were designed, synthesized and evaluated as human chymase inhibitors. Structure-activity relationship studies led to the identification of a potent inhibitor, (6S)-6-(5-chloro-2-methoxybenzyl)-4-[(4-chlorophenyl)sulfonyl]-1,4-diazepane-2,5-dione, with an IC(50) of 0.027 microM.     

Selective inhibition of herpes simplex virus by 5-amino-2,5-dideoxy-5-iodouridine.

5-Amino-2,5-dideoxy-5-iodouridine, a nel thymidine analogue, is a potent inhibitor of herpes simplex virus type 1 replication. In contrast to most other nucleoside analogues which possess antiviral activity, 5-amino-2,5-dideoxy-5-iodouridine exhibits little, if any, cellular toxicity. Preliminary evidence suggests that 5-amino-2,5-dideoxy-5-iodouridine selectively inhibits viral-specific DNA synthesis.     

Derivatives of 2,5-anhydroallitol and 2,5-anhydroaltritol

Two routes to protected derivatives of 2,5-anhydroallitol were investigated. The first route, involving a two-step reduction of 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-allonitrile (4), gave a mixture of 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-altritol (7) and a lesser amount of the desired 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-allitol (6). Isomerization was shown to occur in the first reduction step—treatment of the nitrile 4 with Raney nickel, sodium hypophosphite, and acetic acid. The second route gave isomerically pure 2,5-anhydro-3,4,6-tri-O-benzyl-D-allitol (21) via reduction of the corresponding ethyl allonate (18).     

Synthesis and evaluation of 2,5-diamino and 2,5-dianilinomethyl pyridine analogues as potential CXCR4 antagonists

CXCR4 and its cognate ligand CXCL12 has been linked to various pathways such as cancer metastasis, inflammation, HIV-1 proliferation, and auto-immune diseases. Small molecules have shown potential as CXCR4 inhibitors and modulators, and therefore can mitigate diseases related to the CXCR4-CXCL12 pathway. We have designed and synthesized a series of 2,5-diamino and 2,5-dianilinomethyl pyridine derivatives as potential CXCR4 antagonists. Thirteen compounds have an effective concentration (EC) of 100?nM or less in a binding affinity assay and nine of these have at least 75% inhibition of invasion in Matrigel binding assay. Compounds 3l, 7f, 7j, and 7p show a minimal reduction in inflammation when carrageenan paw edema test is conducted. Overall, these compounds show potential as CXCR4 antagonist.     

Synthesis and biological evaluation of 2,5-dihydropyrazol

A novel series of 2,5-dihydropyrazolo[4,3-c]quinolin-3-ones has been prepared. These compounds showed good PDE 4 inhibitory activity and weak affinity for rolipram's binding site. They also exhibited a good anti-inflammatory profile without emetic side effects.     

Urinary 2,5 hexanedione as a biomarker of n-hexane exposure

n-Hexane is a saturated aliphatic hydrocarbon widely used in industry. In most cases it is used as a mixture with hexane isomers and various others solvents in the form of commercial hexane. n-Hexane is metabolized oxidatively to a number of compounds, including 2,5-hexanedione (2,5-HD), which is eliminated through the urine and is implicated in the neurotoxic effect of this solvent. The main objective of this study was to evaluate urinary 2,5-HD as a biomarker of n-hexane exposure. The study was carried out in seven industrial units. Post-shift urine samples from 111 workers who handled commercial hexane were collected and analysed for 2,5-HD by capillary gas chromatography. Air sampling was performed in the breathing zones of the workers, and the air samples were analysed using validated methods. Monitoring individual exposures showed that n-hexane exposure varied from 5 to 70 p.p.m. (mean±SD = 15.24±2.98 p.p.m.). Significant correlation was observed between exposure to n-hexane and urinary 2,5-HD levels, with high correlation coefficients (ρ= 0.81, p = 0.000), suggesting that urinary 2,5-HD is a good biomarker of occupational exposure to n-hexane. Urinary 2,5-HD is recommended as a better tool than air monitoring in the assessment of health risk, namely the early detection of n-hexane neurotoxicity.     

Urinary 2,5 hexanedione as a biomarker of n-hexane exposure

n-Hexane is a saturated aliphatic hydrocarbon widely used in industry. In most cases it is used as a mixture with hexane isomers and various others solvents in the form of commercial hexane. n-Hexane is metabolized oxidatively to a number of compounds, including 2,5-hexanedione (2,5-HD), which is eliminated through the urine and is implicated in the neurotoxic effect of this solvent. The main objective of this study was to evaluate urinary 2,5-HD as a biomarker of n-hexane exposure. The study was carried out in seven industrial units. Post-shift urine samples from 111 workers who handled commercial hexane were collected and analysed for 2,5-HD by capillary gas chromatography. Air sampling was performed in the breathing zones of the workers, and the air samples were analysed using validated methods. Monitoring individual exposures showed that n-hexane exposure varied from 5 to 70 p.p.m. (mean ±SD = 15.24 ±2.98 p.p.m.). Significant correlation was observed between exposure to n-hexane and urinary 2,5-HD levels, with high correlation coefficients ( ρ= 0.81, p = 0.000), suggesting that urinary 2,5-HD is a good biomarker of occupational exposure to n-hexane. Urinary 2,5-HD is recommended as a better tool than air monitoring in the assessment of health risk, namely the early detection of n-hexane neurotoxicity.     

3,4-Diamino-2,5-thiadiazole-1-oxides as potent CXCR2/CXCR1 antagonists

A series of novel and potent 3,4-diamino-2,5-thiadiazole-1-oxides were prepared and found to show excellent binding affinities for CXCR2 and CXCR1 receptors and excellent inhibitory activity of Gro-alpha and IL-8 mediated in vitro hPMN MPO release of CXCR2 and CXCR1 expressing cell lines. On the other hand, a closely related 3,4-diamino-2,5-thiadiazole-dioxide did not show functional activity despite its excellent binding affinities for CXCR2 and CXCR1 in membrane binding assays. A detailed SAR has been discussed in these two closely related structures.     

Asymmetric Trans-esterification of meso-2,5-Dibromoadipate and Synthesis of Optically Active cis-2,5-Disubstituted Pyrrolidines

Asymmetric trans-esterification of meso-2,5-dibromoadipate to (–)-benzyl methyl 2,5-dibromoadipate by lipase with subsequent chemical reactions afforded optically active cis-2,5-disubstituted pyrrolidines. An equivalent asymmetric transformation was performed by selectively hydrolyzing a cis-2,5-disubstituted pyrrolidine having a chiral N-substituent.     

2,5-Dideoxy-2,5-imino-d-altritol as a new class of pharmacological chaperone for Fabry disease

Chromatographic separation of the extract from roots of Adenophora triphylla resulted in the isolation of two pyrrolidines, six piperidines, and two piperidine glycosides. The structures of new iminosugars were elucidated by spectroscopic methods as 2,5-dideoxy-2,5-imino-d-altritol (DIA) (2), β-1-C-butenyl-1-deoxygalactonojirimycin (8), 2,3-dideoxy-β-1-C-ethyl-1-deoxygalactonojirimycin (9), and 6-O-β-d-glucopyranosyl-2,3-dideoxy-β-1-C-ethyl-1-deoxygalactonojirimycin (10). β-1-C-Butyl-1-deoxygalactonojirimycin (7) and compound 8 were found to be better inhibitors of α-galactosidase than N-butyl-1-deoxygalactonojirimycin. The present work elucidated that DIA was a powerful competitive inhibitor of human lysosome α-galactosidase A (α-Gal A) with a K_i value of 0.5 μM. Furthermore, DIA improved the thermostability of α-Gal A in vitro and increased intracellular α-Gal A activity by 9.6-fold in Fabry R301Q lymphoblasts after incubation for 3 days. These experimental results suggested that DIA would act as a specific pharmacological chaperone to promote the smooth escape from the endoplasmic reticulum (ER) quality control system and to accelerate transport and maturation of the mutant enzyme.     

Evidence for a global inhibitor-induced conformation change on the Ca(2+)-ATPase of sarcoplasmic reticulum from paired inhibitor studies

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by a variety of hydrophobic, hydroxy-containing molecules. A kinetic method has been used to study competition between binding of pairs of inhibitors to the ATPase. The presence of 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ) decreases the affinity of the ATPase for 2,5-dipropyl-1,4-dihydroxybenzene (PHQ), suggesting that PHQ and BHQ bind to the same site on the ATPase. In contrast, the presence of BHQ increases the affinity of the ATPase for curcumin and vice versa. This suggests that BHQ and curcumin bind to separate sites on the ATPase and that binding of the first inhibitor to the ATPase results in a change to a conformation with higher affinity for the second inhibitor. This is consistent with previous experiments with BHQ and thapsigargin suggesting a conformation change on inhibitor binding, E2 + I <--> 2; E2I <--> 2; E2(A)I, with E2(A)I having a higher affinity for the second inhibitor than E2. The affinity for BHQ is also increased by binding of diethylstilbesterol, ellagic acid, or nonylphenol, and the affinity for curcumin is also increased by ellagic acid. These results showing that binding of a variety of inhibitors of very different structures all result in a general increase in inhibitor affinity point to a global conformational change on the Ca(2+)-ATPase caused by inhibitor binding, as well as any local, inhibitor-specific changes in conformation.     

Bioactivation of the fungal phytotoxin 2,5-anhydro-D-glucitol by glycolytic enzymes is an essential component of its mechanism of action

An isolate of Fusarium solani, NRRL 18883, produces the natural phytotoxin 2,5-anhydro-D-glucitol (AhG). This fungal metabolite inhibited the growth of roots (150 of 1.6 mM), but it did not have any in vitro inhibitory activity. The mechanism of action of AhG requires enzymatic phosphorylation by plant glycolytic kinases to yield AhG-1,6-bisphosphate (AhG-1,6-bisP), an inhibitor of Fru-1,6-bisP aldolase. AhG-1,6-bisP had an I50 value of 570 microM on aldolase activity, and it competed with Fru-1,6-bisP for the catalytic site on the enzyme, with a Ki value of 103 microm. The hydroxyl group on the anomeric carbon of Fru-1,6-bisP is required for the formation of an essential covalent bond to zeta amino functionality of lysine 225. The absence of this hydroxyl group on AhG-1,6-bisP prevents the normal catalytic function of aldolase. Nonetheless, modeling of the binding of AhG-1,6-bisP to the catalytic pocket shows that the inhibitor interacts with the amino acid residues of the binding site in a manner similar to that of Fru-1,6-bisP. The ability of F. solani to produce a fructose analog that is bioactivated by enzymes of the host plant in order to inhibit a major metabolic pathway illustrates the intricate biochemical processes involved in plant-pathogen interactions.     

Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases

2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68-75.     

Degradation of 2,5-dimethylpyrazine by <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> strain DP-45 isolated from a waste gas treatment plant of a fishmeal processing company

A bacterium, strain DP-45, capable of degrading 2,5-dimethylpyrazine (2,5-DMP) was isolated and identified as Rhodococcus erythropolis. The strain also grew on many other pyrazines found in the waste gases of food industries, like 2,3-dimethylpyrazine (2,3-DMP), 2,6-dimethylpyrazine (2,6-DMP), 2-ethyl-5(6)-dimethylpyrazine (EMP), 2-ethylpyrazine (EP), 2-methylpyrazine (MP), and 2,3,5-trimethylpyrazine (TMP). The strain utilized 2,5-DMP as sole source of carbon and nitrogen and grew optimally at 25°C with a doubling time of 7.6 h. The degradation of 2,5-DMP was accompanied by the growth of the strain and by the accumulation of a first intermediate, identified as 2-hydroxy-3,6-dimethylpyrazine (HDMP). The disappearance of HDMP was accompanied by the release of ammonium into the medium. No other metabolite was detected. The degradation of 2,5-DMP and HDMP by strain DP-45 required molecular oxygen. The expression of the first enzyme in the pathway was induced by 2,5-DMP and HDMP whereas the second enzyme was constitutively expressed. The activity of the first enzyme was inhibited by diphenyliodonium (DPI), a flavoprotein inhibitor, methimazole, a competitive inhibitor of flavin-containing monooxygenases, and by cytochrome P450 inhibitors, 1-aminobenzotriazole (ABT) and phenylhydrazine (PHZ). The activity of the second enzyme was inhibited by DPI, ABT, and PHZ. Sodium tungstate, a specific antagonist of molybdate, had no influence on growth and consumption of 2,5-DMP by strain DP-45. These results led us to propose that a flavin-dependent monooxygenase or a cytochrome P450-dependent monooxygenase rather than a molybdenum hydroxylase catalyzed the initial hydroxylation step and that a cytochrome P450 enzyme is responsible for the transformation of HDMP in the second step.