Structural and biochemical analyses of concanavalin A circular permutation by jack bean asparaginyl endopeptidase.

Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.

Synthesis and anti-ureolitic activity of Biginelli adducts derived from formylphenyl boronic acids.

Urease is a metalloenzyme that contains two Ni(II) ions in its active site and catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The development of effective urease inhibitors is crucial not only for mitigating nitrogen losses in agriculture but also for offering an alternative treatment against infections caused by resistant pathogens that utilize urease as a virulence factor. This study focuses on synthesizing and investigating the urease inhibition potential of Biginelli Adducts bearing a boric acid group. An unsubstituted or hydroxy-substituted boronic group in the Biginelli adducts structure enhances the urease inhibitory activity. Biophysical and kinetics studies revealed that the best Biginelli adduct (4e; IC50 = 132 ± 12 µmol/L) is a mixed inhibitor with higher affinity to the urease active site over an allosteric one. Docking studies confirm the interactions of 4e with residues essential for urease activity and demonstrate its potential to coordinate with the nickel atoms through the oxygen atoms of carbonyl or boronic acid groups. Overall, the Biginelli adduct 4e shows great potential as an additive for developing enhanced efficiency fertilizers and/or for medical applications.

Designing and Synthesis of Novel Fexofenadine-Derived Hydrazone-Schiff Bases as Potential Urease Inhibitors: In-Vitro, Molecular Docking and DFT Investigations.

Thirteen novel hydrazone-Schiff bases (3-15) of fexofenadine were succesfully synthesized, structurally deduced and finally assessed their capability to inhibit urease enzyme (in vitro). In the series, six compounds 12 (IC50=10.19±0.16 µM), 11 (IC50=15.05±1.11 µM), 10 (IC50=17.01±1.23 µM), 9 (IC50=17.22±0.81 µM), 13 (IC50=19.31±0.18 µM), and 14 (IC50=19.62±0.21 µM) displayed strong inhibitory action better than the standard thiourea (IC50=21.14±0.24 µM), while the remaining compounds displayed significant to less inhibition. LUMO and HOMO showed the transferring of charges from molecules to biological transfer and MEP map showed the chemically reactive zone appropriate for drug action are calculated using DFT. AIM charges, non-bonding orbitals, and ELF are also computed. The urease protein binding analysis benefited from the docking studies.

Green synthesis of silver and gold nanoparticles using Crataegus oxyacantha extract and their urease inhibitory activities.

This study reports the green synthesis and urease inhibitory activities of Ag and Au nanoparticles (NPs) using Crataegus oxyacantha extract. The synthesized NPs were characterized by UV-visible, FT-IR spectroscopy, atomic force microscopy, and scanning electron microscopy. The obtained NPs were spherical in shape, and their size was around 85 nm. A strong correlation between the phytochemicals present in the extract and their capability for the synthesis of NPs was observed. Furthermore, the shape, size, stability, and bioactivity of the NPs were strongly influenced by the stabilizing phytochemicals. The experimental analysis suggested that these NPs have substantial stability in a diverse range of physiological conditions such as pH, salinity, and temperature. The NPs exhibited potent urease enzyme inhibitory activities with percent inhibition of 99.25 and IC50 value of 1.38 ± 0.3, comparable to the standard (thiourea percent inhibition, that is, 98.2% and IC50 value 5.3 ± 0.04). These results suggested that the proposed NPs could be used in the homeopathic and pharmaceutical industries for biomedical applications.

Hydrazine clubbed 1,3-thiazoles as potent urease inhibitors: design, synthesis and molecular docking studies.

Synthesis of a novel series of hydrazine clubbed 1,3-thiazoles (5a-m) has been described by reacting hydrazine-1-carbothioamides (3a-k) with α-chloro- or bromo-acetophenones (4a-d) in refluxing ethanol in good to excellent yields (65-86%). Structural confirmation was based upon spectroscopic techniques such as 1H-NMR, 13C-NMR, FT-IR and mass spectrometry. The biological application of these motifs has been demonstrated in terms of their strong urease inhibition activity. The results of in vitro study revealed that all the compounds are the potent inhibitors of urease. The IC50 (ranging in between 110 and 440 nM) values were higher as compared to that of standard, i.e., thiourea (IC50 = 490 ± 10 nM). The synthesized compounds were docked at the active sites of the Jack bean urease enzyme in order to explore the possible binding interactions of enzyme-ligand complexes; the results reinforced the in vitro biological activity results.

Hybrid Multivalent Jack Bean α-Mannosidase Inhibitors: The First Example of Gold Nanoparticles Decorated with Deoxynojirimycin Inhitopes.

Among carbohydrate-processing enzymes, Jack bean α-mannosidase (JBα-man) is the glycosidase with the best responsiveness to the multivalent presentation of iminosugar inhitopes. We report, in this work, the preparation of water dispersible gold nanoparticles simultaneously coated with the iminosugar deoxynojirimycin (DNJ) inhitope and simple monosaccharides (ß-d-gluco- or α-d-mannosides). The display of DNJ at the gold surface has been modulated (i) by using an amphiphilic linker longer than the aliphatic chain used for the monosaccharides and (ii) by presenting the inhitope, not only in monomeric form, but also in a trimeric fashion through combination of a dendron approach with glyconanotechnology. The latter strategy resulted in a strong enhancement of the inhibitory activity towards JBα-man, with a Ki in the nanomolar range (Ki = 84 nM), i.e., more than three orders of magnitude higher than the monovalent reference compound.

Probing sulphamethazine and sulphamethoxazole based Schiff bases as urease inhibitors; synthesis, characterization, molecular docking and ADME evaluation.

In the current study, a novel series of Schiff base derivatives of (E)-4-(benzylideneamino)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (3a-3f) and (E)-4-(benzylideneamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (3g-3q) were synthesize. The structures of synthetic compounds were elucidated by various spectroscopic techniques such as FTIR, NMR and spectrometric HRMS analysis. Synthetic derivatives were evaluated for their Jack Bean urease inhibitory activity using established in-vitro assay. It is worth mentioning here that most of our derivatives of both series displayed moderate to strong inhibitory activity, ranging between IC50 = 2.48 ± 0.78 µM and 35.63 ± 1.26 µM, as compared to standard thiourea (IC50 = 20.03 ± 2.03 µM). Further, structure activity relationship studies suggest that the presence of halogen at ortho and para positions on the aryl ring in (E)-4-(benzylideneamino)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide derivatives and hydroxy and halogen in (E)-4-(benzylideneamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide derivatives increased the urease inhibitory activity. Furthermore, molecular docking studies were carried out in order to investigate the binding mode of this class of compounds to urease. In order to evaluate drug likeness of compounds ADME evaluation was done, and the synthesized compounds were found to be non-toxic and present passive gastrointestinal absorption. The data suggests the synthesized sulphamethazine and sulphamethoxazole derivatives can serve as a novel scaffold to inhibit urease.

Flavonoid analogues as urease inhibitors: Synthesis, biological evaluation, molecular docking studies and in-silico ADME evaluation.

A series of novel flavonoid analogues were designed and synthesized. The aimed compounds for urease inhibitory activities were clearly superior to the control drug thiourea (more than 10 times). Among these compounds, L2 (IC50 = 1.343 µM) and L12 (IC50 = 1.207 µM) exhibited the most excellent urease inhibitory activity in vitro. The molecular dockings of L2, L12 and L22 into urease were performed to explore the binding modes and their structure-activity relationship. Furthermore, these aimed compounds showed good druggable properties.

Synthesis, characterization and molecular docking of some novel hydrazonothiazolines as urease inhibitors.

A series of new hydrazonothiazolines (3a-v) was obtained in good to excellent yields (79-96%) via cyclization of the appropriate thiosemicarbazones with phenacyl bromide. The targeted compounds were characterized by advanced spectroscopic techniques, such as FTIR, 1HNMR, 13CNMR and ESI-MS. The structure of compounds 3n and 3v was unambiguously confirmed by single crystal X-ray analysis. All compounds displayed enhanced inhibitory activity against urease enzyme with IC50 values in range of 1.73 ±â€¯1.57-27.3 ±â€¯0.655 µM when compared to standard thiourea (IC50 = 20.8 ±â€¯0.75 µM). The structure-activity relationship studies demonstrated that the activity of this series is due the central thiazole ring that interacts with nickel atoms in the active site of urease enzyme. Moreover, molecular docking studies were carried out to investigate the binding mode of all active compounds and an inactive (3u) with the active site of the urease enzyme. The docking results are in complete agreement with the experimental finding.

Robust therapeutic potential of carbazole-triazine hybrids as a new class of urease inhibitors: A distinctive combination of nitrogen-containing heterocycles.

The inhibition of urease enzyme is very important as it plays a key role in the treatment of several urinary and gastrointestinal tract infections. This enzyme provides a suitable environment for Helicobacter pylori at the low pH of the stomach, a causative agent of gastric and peptic ulcer that may lead to cancer. In agriculture, the high urease content causes environmental and economic problems. In this pursuit, given the well-established importance of integrated pharmacophores in medicinal chemistry and to explore new inhibitors of urease featuring two distinct heterocyclic functionalities, we herein report a facile synthesis of carbazole-triazine hybrids (3a-j). These new propeller-shaped chemical scaffolds were evaluated for their urease inhibitory potential in order to identify suitable leads. The initial structure-activity survey work guided through in vitro bioactivity results recognized 3e and 3f as new starting point hits incorporating bulky iodo (3e) and strong electron-withdrawing nitro (3f) groups at the para-position of aryl amine component. The potent compounds (3e &3f) exhibited the highest activity with IC50 values of 5.6 and 6.7 µM, respectively. In the molecular docking analysis, these compounds depicted excellent binding interactions with the active site residues. The key interactions observed include hydrogen bonding, π-π interactions, π-cation and nickel atom coordination to the triazine nitrogen of both inhibitors.

Synthesis, Biological Evaluation and Molecular Docking of Deferasirox and Substituted 1,2,4-Triazole Derivatives as Novel Potent Urease Inhibitors: Proposing Repositioning Candidate.

A series of new deferasirox derivatives were synthesized through the reaction of monosubstituted hydrazides with 2-(2-hydroxyphenyl)-4H-benzo[e][1,3]oxazin-4-one. For the first time, deferasirox and some of its derivatives were evaluated for their in vitro inhibitory activity against Jack bean urease. The potencies of the members of this class of compounds are higher than that of acetohydroxamic acid. Two compounds, bearing tetrazole and hydrazine derivatives (bioisoester of carboxylate group), represented the most potent urease inhibitory activity with IC50 values of 1.268 and 3.254 µm, respectively. In silico docking studies were performed to delineate possible binding modes of the compounds with the enzyme, urease. Docking analysis suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket and thus restrict the mobility of the flap by interacting with its crucial amino acid residues, CME592 and His593. The overall results of urease inhibition have shown that these target compounds can be further optimized and developed as a lead skeleton for the discovery of novel urease inhibitors.

Cross-linkage urease nanoparticles: a high-efficiency signal-generation tag for portable pH meter-based electrochemical immunoassay of lipocalin-2 protein diagnostics.

An innovative signal-transduction tag based on cross-linked urease nanoparticles (CLENP) was designed for the development of a pH meter-based immunoassay of lipocalin-2 (LCN2). The CLENP was synthesized with a typical desolvation method using ethanol as desolvation agent, followed by functionalization with polyaspartic acid. The carboxylated CLENP were used as the signal-generation tags for the labelling of secondary antibodies via the carbodiimide coupling. Upon target LCN2 introduction, a sandwich-type immune reaction was performed between capture antibody-coated plate and the labeled secondary antibody on the CLENP. The conjugated CLENP in the microplate hydrolyzed urea into ammonia (NH4+) and carbonate (CO32-), resulting in the pH change of solution, which was determined with a handheld pH meter. The pH variation was proportional to target concentration in the sample. By monitoring the pH variation of the urea solution, the level of LCN2 at a concentration as low as 5.2 pg mL-1 was evaluated. The pH meter-based electrochemical immunoassay can be utilized for mass production of miniaturized lab-on-a-chip devices with handheld pH meter, thereby opening new opportunities for protein diagnostics and biosecurity. Graphical abstract An innovative signal-transduction tag based on cross-linked urease nanoparticles was designed for high-efficiency immunoassay of lipocalin-2 with pH meter readout.

Electrical Biosensing at Physiological Ionic Strength Using Graphene Field-Effect Transistor in Femtoliter Microdroplet.

Graphene has strong potential for electrical biosensing owing to its two-dimensional nature and high carrier mobility which transduce the direct contact of a detection target with a graphene channel to a large conductivity change in a graphene field-effect transistor (G-FET). However, the measurable range from the graphene surface is highly restricted by Debye screening, whose characteristic length is less than 1 nm at physiological ionic strength. Here, we demonstrated electrical biosensing utilizing the enzymatic products of the target. We achieved quantitative measurements of a target based on the site-binding model and real-time measurement of the enzyme kinetics in femtoliter microdroplets. The combination of a G-FET and microfluidics, named a "lab-on-a-graphene-FET", detected the enzyme urease with high sensitivity in the zeptomole range in 100 mM sodium phosphate buffer. Also, the lab-on-a-graphene-FET detected the gastric cancer pathogen Helicobacter pylori captured at a distance greater than the Debye screening length from the G-FET.

Periodic Nucleation of Calcium Phosphate in a Stirred Biocatalytic Reaction.

Highly ordered superstructures composed of inorganic nanoparticles appear in natural and synthetic systems, however the mechanisms of non-equilibrium self-organization that may be involved are still poorly understood. Herein, we performed a kinetic investigation of the precipitation of calcium phosphate using a process widely found in microorganisms: the hydrolysis of urea by enzyme urease. With high initial ratio of calcium ion to phosphate, periodic precipitation was obtained accompanied by pH oscillations in a well-stirred, closed reactor. We propose that an internal pH-regulated change in the concentration of phosphate ion is the driving force for periodicity. A simple model involving the biocatalytic reaction network coupled with burst nucleation of nanoparticles above a critical supersaturation reproduced key features of the experiments. These findings may provide insight to the self-organization of nanoparticles in biomineralization and improve design strategies of biomaterials for medical applications.

Enhanced Diffusion and Oligomeric Enzyme Dissociation.

The concept that catalytic enzymes can act as molecular machines transducing chemical activity into motion has conceptual and experimental support, but experimental support has involved oligomeric enzymes, often studied under conditions where the substrate concentration is higher than biologically relevant and accordingly exceeds kM, the Michaelis constant. Urease, a hexamer of subunits, has been considered to be the gold standard demonstrating enhanced diffusion. Here we show that urease and certain other oligomeric enzymes dissociate above kM into their subunits that diffuse more rapidly, thus providing a simple physical mechanism that contributes to enhanced diffusion in this regime of concentrations. Mindful that this conclusion may be controversial, our findings are supported by four independent analytical techniques: static light scattering, dynamic light scattering (DLS), size-exclusion chromatography (SEC), and fluorescence correlation spectroscopy (FCS). Data for urease are emphasized and the conclusion is validated for hexokinase, acetylcholinesterase, and aldolase. For hexokinase and aldolase no enhanced diffusion is observed except under conditions when these oligomeric enzymes dissociate. At substrate concentration regimes below kM at which acetylcholinesterase and urease do not dissociate, our finding showing up to 10% enhancement of the diffusion coefficient is consistent with various theoretical scenarios in the literature.

Not-XOR (NXOR) Logic Gate Realized with Enzyme-Catalyzed Reactions: Optical and Electrochemical Signal Transduction.

The studied enzyme-based biocatalytic system mimics NXOR Boolean logic gate, which is a logical operator that corresponds to equality in Boolean algebra. It gives the functional value true (1) if both functional arguments (input signals) have the same logical value (0,0 or 1,1), and false (0) if they are different (0,1 or 1,0). The output signal producing reaction is catalyzed by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH), which is inhibited at acidic and basic pH values. Two other reactions catalyzed by esterase and urease produce acetic acid and ammonium hydroxide, respectively, shifting solution pH from the optimum pH for PQQ-GDH to acidic and basic values (1,0 and 0,1 input combinations, respectively), thus switching the enzyme activity off (output 0). When the input signals are not applied (0,0 combination) or both applied compensating each other (1,1 combination) the optimum pH is preserved, thus keeping PQQ-GDH running at the high rate (output 1). The biocatalytic cascade mimicking the NXOR gate was characterized optically and electrochemically. In the electrochemical experiments the PQQ-GDH enzyme communicated electronically with a conducting electrode support, thus resulting in the electrocatalytic current when signal combinations 0,0 and 1,1 were applied. The logic gate operation, when it was realized electrochemically, was also extended to the biomolecular release controlled by the gate. The release system included two electrodes, one performing the NXOR gate and another one activated for the release upon electrochemically stimulated alginate hydrogel dissolution. The studied system represents a general approach to the biocatalytic realization of the NXOR logic gate, which can be included in different catalytic cascades mimicking operation of concatenated gates in sophisticated logic circuitries.

Investigation on the effect of alkyl chain linked mono-thioureas as Jack bean urease inhibitors, SAR, pharmacokinetics ADMET parameters and molecular docking studies.

The increasing resistance of pathogens to common antibiotics, as well as the need to control urease activity to improve the yield of soil nitrogen fertilization in agricultural applications, has stimulated the development of novel classes of molecules that target urease as an enzyme. In this context, the newly developed compounds on the basis of 1-heptanoyl-3-arylthiourea family were evaluated for Jack bean urease enzyme inhibition activity to validate their role as potent inhibitors of this enzyme. 1-Heptanoyl-3-arylthioureas were obtained in excellent yield and characterized through spectral and elemental analysis. All the compounds displayed remarkable potency against urease inhibition as compared to thiourea standard. It was found that novel compounds fulfill the criteria of drug-likeness by obeying Lipinski's rule of five. Particularly compound 4a and 4c can serve as lead molecules in 4D (drug designing discovery and development). Kinetic mechanism and molecular docking studies also carried out to delineate the mode of inhibition and binding affinity of the molecules.

Synthesis, crystal structure and biological evaluation of new phosphoramide derivatives as urease inhibitors using docking, QSAR and kinetic studies.

In an attempt to achieve a new class of phosphoramide inhibitors with high potency and resistance to the hydrolysis process against urease enzyme, we synthesized a series of bisphosphoramide derivatives (01-43) and characterized them by various spectroscopic techniques. The crystal structures of compounds 22 and 26 were investigated using X-ray crystallography. The inhibitory activities of the compounds were evaluated against the jack bean urease and were compared to monophosphoramide derivatives and other known standard inhibitors. The compounds containing aromatic amines and their substituted derivatives exhibited very high inhibitory activity in the range of IC50 = 3.4-1.91 × 10-10 nM compared with monophosphoramides, thiourea, and acetohydroxamic acid. It was also found that derivatives with PO functional groups have higher anti-urease activity than those with PS functional groups. Kinetics and docking studies were carried out to explore the binding mechanism that showed these compounds follow a mixed-type mechanism and, due to their extended structures, can cover the entire binding pocket of the enzyme, reducing the formation of the enzyme-substrate complex. The quantitative structure-activity relationship (QSAR) analysis also revealed that the interaction between the enzyme and inhibitor is significantly influenced by aromatic rings and PO functional groups. Collectively, the data obtained from experimental and theoretical studies indicated that these compounds can be developed as appropriate candidates for urease inhibitors in this field.

Synthesis and characterization of new thiosemicarbazones, as potent urease inhibitors: In vitro and in silico studies.

A new series of N-substituted thiosemicarbazones (3a-u) bearing 2-naphthyl and dihydrobenzofuranyl scaffolds were synthesized in good to excellent yields (78-95%). The synthesized compounds were characterized by advanced spectroscopic techniques, such as FTIR, 1HNMR, 13CNMR and ESI-MS and evaluated as urease inhibitors. The structure of compound 3m was unambiguously confirmed by single crystal X-ray analysis. All compounds showed remarkable activities against urease enzyme with IC50 values in range of 1.4-36.1 µM. The majority of the synthesized compounds showed higher activity than the standard compound thiourea. Molecular docking was performed to study the mode of interaction of these compounds and their structure-activity relationship. These studies revealed that the compounds bind at the active site and interacts with the nickel atom present in the binding site. The molecular docking demonstrated excellent co-relations with the experimental findings.

Isoindolin-1-one derivatives as urease inhibitors: Design, synthesis, biological evaluation, molecular docking and in-silico ADME evaluation.

An efficient, one-pot and four-component synthesis of a new series of 2,3-disubstituted isoindolin-1-ones is described and their Jack bean urease inhibitory activities are evaluated. Heating a mixture of 1,1-bis(methylthio)-2-nitroethene, a 1,2-diamine, a 2-formylbenzoic acid and a primary amine in EtOH for 3.5 h afforded the corresponding 2,3-disubstituted isoindolin-1-ones in good to excellent yields. All sixteen synthesized isoindolin-1-one derivatives 5a-p showed urease inhibitory activity. Among them, 5c showed the most urease inhibitory activity (IC50 = 10.07 ±â€¯0.28 µM) being over 2-fold more potent than thiourea (IC50 = 22.01 ±â€¯0.10 µM) and 10-fold than hydroxyurea (IC50 = 100.00 ±â€¯0.02 µM) as the standard inhibitors, respectively. Also, results from molecular docking studies were in good agreement with those obtained from in vitro tests.