Urease Inhibitors Research Articles

Effect of seaweed extracts from different sources combined with urease and nitrification inhibitors

Urease inhibitors (UIs) and nitrification inhibitors (NIs) still have limitations in increasing crop yield. Therefore, to improve the application effect of inhibitors, the combination of seaweed extracts (SE) from different sources and inhibitors was added to urea to provide a theoretical basis for the development of a new generation of efficient stabilized urea fertilizer with both biostimulant and inhibitor technologies. The combinations were tested in outdoor pots with no N- fertilizer (CK), application of urea alone (U) as control, and kelp polysaccharide (KP), margin polysaccharide (MP), N−(n−propyl) thiophosphoric triamide (NPPT), dicyandiamide (DCD), and combinations of SE with inhibitor were added to urea to make eight fertilizer prototypes. Compared with KP, MP showed better application effect, with significantly higher grain yield and nitrogen use efficiency (NUE) (P < 0.05). Compared with the addition of inhibitor alone, the combinations of NPPT with KP and MP, respectively, had opposite effects on urea−N transformation, meanwhile NPPT+KP had a positive effect. However, NPPT+MP significantly decreased yield, plant nitrogen uptake, and NUE (P < 0.05); DCD+MP decreased plant N uptake and NUE to some extent. Therefore, the addition of NPPT with KP and DCD with KP to urea significantly improved yield when planting maize in black soil.

Change in maize yield, N use efficiencies and climatic warming potential after urea combined with Nitrapyrin and NBPT during the growing season in a black soil

Increasing nitrogen (N) utilization and reducing N losses are a globally accepted strategy for N fertilizer application. However, little information has been reported on the fate of N when urea (U) and nitrification and/or urease inhibitors are used in black soils. Therefore, a two-year field experiment was conducted with three N fertilizer application treatments, including urea-only, urea combined with nitrification inhibitor (U+NI), urea combined with NI and urease inhibitor (UI) treatments. Maize (Zea mays L.) yield, N use efficiency, gas emissions, global warming potential (GWP), and apparent N loss were determined. Compared to the other treatments, the U+NI+UI treatment had the significantly (P < 0.05) highest maize yield (11.2 t ha-1), N recovery efficiency (44.5%), agronomic N efficiency (28.7 kg kg-1) and N partial factor productivity (75.8 kg kg-1). Moreover, the U+NI+UI treatment significantly (P < 0.05) reduced the peak of Ammonia (NH3) volatilization and nitrous oxide (N2O) emissions, cumulative NH3 volatilization and N2O emissions, and apparent N loss compared to the U treatment. Although there was no significant difference in cumulative N2O emissions between the U+NI+UI and U+NI treatments, the U+NI+UI treatment significantly (P < 0.05) reduced cumulative NH3 volatilization by 55.3%, GWP by 16.2%, and apparent N loss by 28.2% compared to the U+NI treatment. Overall, the combination of urea with urease and inhibitors is a good practice to improve maize yield and N use efficiency while reducing environmental impacts in black soil.

Novel benzoxazole-based thiosemicarbazide derivatives as new inhibitors of urease and β-Glucuronidase: Synthesis, in vivo anti-nematodal activity and ADMET prediction along with in silico study

Here, we discuss the synthesis of thiosemicarbazide derivatives based on benzoxazole. These compounds were obtained via sequence of reactions. The targeted products wereconfirmed using a number of spectroscopic methods, including NMR (1H and 13C) and EI-MS. After spectral confirmation all the synthesized compounds were evaluated for urease and β-Glucuronidase inhibitory activity in order to explore their biological significances in the presence of standard drug thiourea (IC50 = 21.86 ± 0.40) and D-saccharic acid 1,4-lactone (IC50 value 22.00 ± 1.10 µM) respectively. Among the evaluated series, compounds 14 and 15 (1.10 and 0.01 and 2.20 and 0.60) were shown to have slightly greater potential than standard drugs. Anti-nematodal activity was also employed to explore the cytotoxic nature of synthesized analogs. In order to establish the binding relationship with enzyme active sites, molecular docking experiments were done and directions for compound modification based on SAR features were addressed. In addition, ADMET prediction study also investigated to found drug like properties of the potential analogs.

Synthesis, Biological Activity Evaluation, Docking and Molecular Dynamics Studies of New Triazole‐Tetrahydropyrimidinone(thione) Hybrid Scaffolds as Urease Inhibitors

New series of triazole-tetrahydropyrimidinone(thione) hybrids (9a-g) were synthesized. FT-IR, 1 H-NMR, 13 C-NMR, elemental analysis and mass spectroscopic studies characterized the structures of the synthesized compounds. Then, the synthesized compounds were screened to determine the urease inhibitory activity. Methyl 4-(4-((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9c) exhibited the highest urease inhibitory activity (IC50 =25.02 μM) among the compounds which was almost similar to thiourea as standard (IC50 =22.32 μM). The docking study of the screened compounds demonstrated that these compounds fit well in the urease active site. Based on the docking study, compound 9c with the highest urease inhibitory activity showed chelates with both Ni2+ ions of the urease active site. Moreover, the molecular dynamic study of the most potent compounds showed that they created important interactions with the active site flap residues, His322, Cys321, and Met317.

Ammonia loss potential and mitigation options in a wheat-maize rotation system in the North China Plain: A data synthesis and field evaluation

The North China Plain (NCP) is an intensive agricultural area and ammonia (NH3) emission hotspot in China. Urgent mitigation actions are needed to reduce its environmental impact. A field trial was conducted, and results combined with a synthesis of existing data to assess the NH3 loss and mitigation potentials in a wheat-maize double-crop rotation, a dominant crop production system in the NCP. Ammonia volatilization losses from N fertilizer applied to summer maize and winter wheat were 9.1% and 5.0%, respectively, based on the data synthesis, and were significantly affected by fertilizer application method and N fertilizer type. From the field trial, optimized reduction in N application rate, compared with conventional practice, had great efficiency for NH3 mitigation. Despite their high NH3 reduction potential, the higher input costs associated with the use of controlled-release fertilizers, replacement of urea with calcium ammonium nitrate or inclusion of double inhibitors (urease and nitrification inhibitors) with urea limited their applicability from an economic perspective. The inclusion of a urease inhibitor with urea achieved over 70% reduction in NH3 emission and also gave a significant improvement in grain yield, net income and N use efficiency. Future adoption of NH3 mitigation options in the NCP should focus on establishing a reduction target and consider the cost-benefits of simultaneous implementation of multiple mitigation techniques.

Urease Inhibition and Structure‐Activity Relationship Study of Thiazolidinone‐, Triazole‐, and Benzothiazole‐Based Heterocyclic Derivatives: A Focus Review

AbstractUrease has a long and distinguished history in the development of enzymology since it was the first enzyme crystallized by Sumner in 1926. The present review article is focused on the urease inhibitory potential of thiazolidinone, triazole, and benzothiazole‐based heterocyclic derivatives. The study begins with the historical developments in the discovery of urease and its substrate, urea, along with the active site architecture of ureases of different origins. The two pathways for the urease‐catalyzed hydrolysis of urea are explained in detail. The urease inhibitory potential of the aforementioned heterocyclic derivatives is reviewed and arranged systematically. Structure‐activity relationships (SARs) study provided the substituents necessary for urease inhibition and will be useful for the researchers working in the field of anti‐ulcer agents. To a step further, important binding interactions were identified with the amino acid residues at the active site of the enzyme. The information gathered is anticipated to offer logical direction and an effective method for creating innovative, potent, and efficient urease inhibitors that will have greater practical uses in the future.

Nitrogen inhibitors improve soil ecosystem multifunctionality by enhancing soil quality and alleviating microbial nitrogen limitation

Soil quality (SQI) is a comprehensive indicator reflecting the agricultural productivity of soil, and soil ecosystem multifunctionality (performing multiple functions simultaneously; EMF) can reflect complex biogeochemical processes. However, the effects of enhanced efficiency nitrogen fertilizers (EENFs; urease inhibitors (NBPT), nitrification inhibitors (DCD), and coated controlled-release urea (RCN)) application on the SQI and soil EMF and their relationships are still unclear. Therefore, we conducted a field experiment to study the effects of different EENFs on the SQI, enzyme stoichiometry and soil EMF in semiarid areas of Northwest China (Gansu, Ningxia, Shaanxi, Shanxi). Across the four study sites, DCD and NBPT increased SQI by 7.61–16.80 % and 2.61 %–23.20 % compared to mineral fertilizer, respectively. N fertilizer application (N200 and EENFs) alleviated microbial N limitation, and EENFs alleviated microbial N and C limitations to a greater extent in Gansu and Shanxi. Moreover, nitrogen inhibitors (Nis; DCD and NBPT) improved the soil EMF to a greater extent than N200 and RCN, DCD increased by 205.82–340.00 % and 145.00–215.47 % in Gansu and Shanxi, respectively; NBPT increased by 332.75–778.59 % and 364.44–929.62 % in Ningxia and Shanxi, respectively. A random forest model showed that the microbial biomass carbon (MBC), microbial biomass nitrogen (MBN) and soil water content (SWC) of the SQI factors were the main driving forces of soil EMF. Moreover, SQI improvement could alleviate microbial C and N limitations and promote the improvement of soil EMF. It is worth noting that soil EMF was mainly affected by microbial N limitation rather than C limitation. Overall, NIs application is an effective way to improve the SQI and soil EMF in the semiarid region of Northwest China.

N-Arylacetamide derivatives of methyl 1,2-benzothiazine-3-carboxylate as potential drug candidates for urease inhibition.

Urease enzyme is an infectious factor that provokes the growth and colonization of virulence pathogenic bacteria in humans. To overcome the deleterious effects of bacterial infections, inhibition of urease enzyme is one of the promising approaches. The current study is designed to synthesize new 1,2-benzothiazine-N-arylacetamide derivatives 5(a-n) that can effectively provide a new drug candidate to avoid bacterial infections by urease inhibition. After structural elucidation by FT-IR, proton and carbon-13 NMR and mass spectroscopy, the synthesized compounds 5(a-n) were investigated to evaluate their inhibitory potential against urease enzyme. In vitro analysis against positive control of thiourea indicated that all the synthesized compounds have strong inhibitory strengths as compared to the reference drug. Compound 5k, being the most potent inhibitor, strongly inhibited the urease enzymes and revealed an IC50 value of 9.8 ± 0.023 µM when compared with the IC50 of thiourea (22.3 ± 0.031 µM)-a far more robust inhibitory potential. Docking studies of 5k within the urease active site revealed various significant interactions such as H-bond, π-alkyl with amino acid residues like Val744, Lys716, Ala16, Glu7452, Ala37 and Asp730.

Combined Effects of Biochar and Inhibitors on Greenhouse Gas Emissions, Global Warming Potential, and Nitrogen Use Efficiency in the Tobacco Field

Biochar (BC), nitrification inhibitors (methyl 3-(4-hydroxyphenyl) propionate, MHPP), and urease inhibitors (n-butyl phosphorothioate triamine, NBPT) have emerged as effective soil greenhouse gas (GHG) mitigation strategies in agroecosystems. However, the combined use of BC and inhibitors in karst areas has no available data. Therefore, the combined effects of BC, MHPP, and NBPT on GHG emissions, global warming potential (GWP) and nitrogen use efficiency (NUE) in roasted tobacco cropping systems were studied to improve the understanding in climate mitigation. CO2, CH4, and N2O emissions from soils were measured using static chamber-gas chromatography. Results showed that the combined use of BC and inhibitors significantly increased soil total nitrogen, available potassium, electric conductivity, pH, and soil organic matter compared to the control. The combined use of BC and MHPP or NBPT significantly increased cumulative soil CO2 emissions by 33.95% and 34.25%, respectively. The exponential–exponential function of soil CO2 fluxes with soil moisture and temperature demonstrated good fit (R2: 0.506–0.836). The combination of BC and NBPT increased the cumulative soil CH4 emissions by 14.28% but not significantly compared to the fertiliser treatment. However, the combination of BC and MHPP resulted in a significant reduction in cumulative soil CH4 emissions by 80.26%. In addition, the combined use of BC and MHPP or NBPT significantly reduced the cumulative soil N2O emissions by 26.55% and 40.67%, respectively. The inhibition effect of NBPT was better than MHPP. Overall, the combined use of BC and inhibitors significantly reduced the yield-scaled GWP, markedly increased crop yield and NUE, and mitigated climate change in the southwest karst region.

Optimization of nitrogen fertilizer of the dark-gray soldized soil of the western forest steppe for using nitrification inhibitor

Nitrogen is a key element for the growth and yield of all crops. Increased doses of nitrogen do not give the expected return, create a danger of leaching of nitrates, activate the emission of nitrous oxide. These problems make it necessary to solve the urgent issue of optimizing nitrogen nutrition using the nitrogen stabilizer nitrapyrin. The purpose of our research under conditions of sufficient moisture in the Western Forest Steppe was to find out the effect of the urease inhibitor on the agrochemical parameters of the dark gray forest podzolized light loam soil, as well as the effect on the yield of winter barley. We conducted field experiments at the Lviv National University of Nature Management. We used traditional methods of field research and standardized methods of laboratory analysis. A positive effect of the use of the nitrogen stabilizer nitrapyrin on the funds of nitrogen, phosphorus and potassium available for nutrition, as well as on the reduction of soil acidity, was established. The highest content of easily hydrolyzable nitrogen was achieved when applying N23P60K60 in autumn + N97 in the phase of vegetation recovery with nitrapyrin – 132 mg/kg of soil. This was 28 mg/kg more than the traditional fertilization system without nitrapyrin (N60P60K60). Application of 120 kg/ha of nitrogen in the form of urea created a reserve 3.3 times higher, and ammonium nitrate created a reserve 3.6 times higher than the unfertilized version. Nitrapyrin, a stabilizer of nitrates, made it possible to limit their formation with the introduction of urea by 3 5%, ammonium nitrate by 10 %. The nitrogen application rate of N120 kg/ha per year led to the annual emission of nitrous oxide in the amount of 121.5 kg/ha. Nitrapyrin limited the amount of nitrous oxide emission by 3.3–7.2 kg/ha, depending on the amount of nitrogen application rate. The use of ammonium nitrate at the rate of N120 when applied in the spring caused the highest level of nitrogen oxide emissions (29.5 kg/ha of soil). However, the application of this fertilizer and inhibitor reduced gaseous nitrogen losses to 25.9 kg/ha when applied to restore vegetation. The introduction of N97 (ammonium nitrate) + N-Lok Max under winter barley during the restoration of vegetation on the background of N23P60K60 under plowing gave the highest average yield in the experiment for 2020–2022 – 7.65 t/ha, for 2022 – 7.90 t/ha. The 3D regression model of winter barley grain yield under the influence of the synergistic effect of both forms of nitrogen illustrates the importance of balancing the funds of forms of this nutrient element in the soil.

Winter wheat responses to enhanced efficiency liquid nitrogen fertilizers in the Canadian Prairies

To evaluate how enhanced efficiency liquid nitrogen (N) fertilizers affect winter wheat ( Triticum aestivum L.) production under irrigated and rain-fed environments, experiments were conducted at two irrigated and five rain-fed sites across the Canadian Prairies from 2013 to 2018 (22 site-years). The N fertilizers included urea ammonium nitrate (UAN) treated with ( i) urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT), ( ii) NBPT plus nitrification inhibitor dicyandiamide, and ( iii) nitrification inhibitor nitrapyrin (Nitrapyrin), as well as untreated UAN and urea, and polymer-coated urea (PCU). All fertilizers were applied by banding 50% at planting and 50% in-crop in early-spring, except PCU, where PCU was applied at planting and urea was applied in early-spring. Nitrous oxide (N2O) emissions and methane (CH4) uptake were measured at one rain-fed site from 2014 to 2017. NBPT increased grain yield by 1.2%–14% and 2.8%–4% under irrigated and rain-fed environments, respectively, relative to all the other N sources except untreated urea in the rain-fed environment. Total N uptake with NBPT was between 0% and 12% higher than the other N sources across irrigated and rain-fed environments. The results suggested that both grain yield and N use efficiency were optimized when UAN contained a urease inhibitor. All liquid enhanced efficiency fertilizers produced grain protein content greater than 11%, except Nitrapyrin under irrigated environments. Data from three site-years indicated that greenhouse gas emissions were unaffected by N source under rain-fed conditions. Liquid UAN with a urease inhibitor may have the most potential to optimize winter wheat production and N use efficiency in the Canadian Prairies.

Winter wheat responses to enhanced efficiency granular nitrogen fertilizer in the Canadian Prairies

Optimizing the timing of nitrogen (N) enhanced efficiency fertilizers (EEFs) may maximize winter wheat ( Triticum aestivum L.) grain yield, protein content, and N-use efficiency (NUE). From 2013 to 2018, experiments were conducted at two irrigated and six rain-fed sites across the Canadian Prairies (24 site-years) to evaluate winter wheat responses to N source and timing/placement effects of EEFs. Nitrogen sources included untreated urea, nitrification inhibitor nitrapyrin treated urea, urease inhibitor N-( n-butyl) thiophosphoric triamide (NBPT) plus nitrification inhibitor dicyandiamide (DCD)-treated urea (NBPT + DCD), and polymer-coated urea (PCU). The N sources were all side-banded at planting, 30% side-banded at planting plus 70% broadcast in-crop late-fall (averaged 38 days after planting; split-applied late-fall), or 30% side-banded at planting plus 70% broadcast in-crop early-spring (averaged 224 days after planting; split-applied early-spring). Nitrous oxide and methane emissions were measured at one rain-fed site to test whether N source and timing/placement influenced CO2-equivalents (CO2-eq; nitrous oxide + methane). Under irrigation, NBPT + DCD consistently produced the highest yields regardless of timing/placement; however, the 80% of the recommended rate caused suboptimal protein responses (≤11%) unless split-application of N was adopted. Untreated urea produced the highest net CO2-eq and yield-scaled CO2-eq emissions, with the highest emissions when urea was split-applied early-spring. To optimize winter wheat production and NUE, we conclude that NBPT + DCD all-banded during seeding operations or split-applied early-spring provided similar and often superior results to other sources, including a more typical system of urea side-banded at the time of seeding.

The Combined Use of Liquid Fertilizer and Urease/Nitrification Inhibitors on Maize Yield, Nitrogen Loss and Utilization in the Mollisol Region

Nitrification inhibitor (NI) and urease inhibitor (UI) with fertilizer have the potential to reduce nitrogen (N) loss as well as improve grain yields. Urea-ammonium nitrate (UAN) solution as liquid fertilizer is superior to conventional solid nitrogen (N) fertilizer in terms of fertilizer efficiency, energy savings, environmental pollution reduction and economic benefits. However, comprehensive assessments of UAN with inhibitors from an environmental and agronomy perspective, including insights into the mechanisms of UAN with inhibitors, are lacking. In a field trial, three single-inhibitor and two double-inhibitor (DI) treatments were set to quantify the grain yield, the N losses and the N recovery efficiency of maize treated with urea supplemented with dicyandiamide (DCD), 3,4-dimethylpyrazole phosphate (DMPP) and N-(n-butyl) thiophosphoric triamide (NBPT). Compared with the UAN treatment, the supply of urease inhibitors reduced NH3 emission by 13.0% but increased N2O emission by 13.0%. The supply of nitrification inhibitors delayed the conversion of ammonium N to nitrate N and improved NH3 emission by 23.5-28.7%, but reduced N2O emission by 31.4% and significantly increased the maize yield by 21.3%. The combined use of NBPT and DCD were not compatible in UAN and cannot achieve the maximum potential for optimizing yields and reducing nitrogen losses. Considering the grain yield, the N use efficiency and the N losses, the combined use of NBPT and DMPP in maize production system significantly improved the grain yield and N use efficiency, as well as reduced N losses.

Different barbiturate derivatives linked to aryl hydrazone moieties as urease inhibitors; design, synthesis, urease inhibitory evaluations, and molecular dynamic simulations

Different barbiturate derivatives linked to aryl hydrazone moieties as urease inhibitors; design, synthesis, urease inhibitory evaluations, and molecular dynamic simulations

Synthesis and biological evaluation of 2-nitrocinnamaldehyde derived thiosemicarbazones as urease inhibitors

Hyperactivity of urease enzyme induces pathogenesis of peptic ulcers and gastritis. The excess of urease can be reduced by introducing various inhibitors. The risk of high urease content also partakes in agriculture field where it declines the environmental health. This alarming situation leads to the mandatory quest of such novel scaffolds that must work wonders in the field of medicinal chemistry. Consequently, we herein report a novel series of N4-substitution of cinnamaldehyde with thiosemicarbazones 3a-q as potential candidates for urease inhibition. These new N4-substituted thiosemicarbazones 3a-q of indistinct chemical scaffolds were characterized by advanced spectroscopic techniques, such as ESI-MS, 13CNMR, 1HNMR, and FTIR. All newly synthesized compounds showed significant urease inhibitions with IC50 values in range of 11.4 ± 0.23 to 80.6 ± 0.52 μM. Furthermore, kinetic studies of compound 3o displayed competitive type of inhibition with ki value 8.65 ± 0.37 µM. For analysis of structure‒activity relationship and interactions pattern of all compounds, molecular docking was used, which revealed excellent binding interactions of compounds with the active site residues, such as nickel atom coordination, π–cation, π–π interactions, and hydrogen bonding. Additionally, the predicted pharmacokinetics and drug-likeness of compounds suggest that these compounds have good pharmacokinetic and physicochemical properties, and these compounds follow rules of drug-likeness.

Kinetic Studies, Antioxidant Activities, Enzyme Inhibition Properties and Molecular Docking of 1,3-Dihydro-1,3-Dioxoisoindole Derivatives

The acid catalyzed hydrolysis of the N-(p-substitutedphenyl) phthalimides in three different acids was investigated at 50.0±0.1°C. Two different antioxidant activity tests as DPPH• and ABTS•+ scavenging activities, and three various enzyme inhibition activity tests as urease, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibition activities, were applied. Compound 3c (2.03 µg/mL ) has higher antioxidant activity than other compounds and standards according to DPPH test. In AChE assay, compounds 3a and 3b (13.13 and 9.59 µg/mL) has higher enzyme inhibition activity than the standard Galantamine (14.37 µg/mL). In BChE and urease tests, all compounds (6.84-13.60 and 10.49-17.73 µg/mL) have higher enzyme inhibition activity than the standard Galantamine (49.40 µg/mL) and thiourea (26.19 µg/mL), respectively. The molecule interaction for each of the three compounds with the active sites of AChE, BChE, and urease enzymes was examined via molecular docking simulations.

Synthesis, Crystal Structures and Urease Inhibition of Zinc Complexes Derived from 4-Chloro-2-(((2-(pyrrolidin-1-yl)ethyl)imino)methyl)phenol.

Three new zinc(II) complexes, [Zn3(μ2-η1:η1-OAc)2(μ2-η2:η0-OAc)2L2] (1), [Zn3(μ2-η1:η1-OAc)2(μ1,1-N3)(N3)L2] (2), [Zn2(μ1,3-N3)(N3)(H2O)L2] (3), with the Schiff base ligand 4-chloro-2-(((2-(pyrrolidin-1-yl)ethyl)imino)methyl)phenol (HL) have been synthesized and characterized by elemental analysis, IR and UV-Vis spectroscopic studies. Crystal structures of the complexes were confirmed by single crystal X-ray diffraction. Complex 1 is a bidentate acetato, monoatomic bridging acetato, and phenolato co-bridged trinuclear zinc compound. The Zn atoms are in octahedral and square pyramidal coordination. Complex 2 is a bidentate acetato, end-on azido, and phenolato co-bridged trinuclear zinc compound. The Zn atoms are in trigonal bipyramidal and square pyramidal coordination. Complex 3 is an end-to-end azido bridged dinuclear zinc compound. The Zn atoms are in square pyramidal and trigonal bipyramidal coordination. The Schiff base ligands in the complexes coordinate to the Zn atoms through the phenolate oxygen, imino nitrogen and pyrrolidine nitrogen. The complexes have interesting inhibitory activity on Jack bean urease, with IC50 values of 7.1-15.3 μmol·L-1.

Unraveling Binding Mechanism and Stability of Urease Inhibitors: A QM/MM MD Study

Soil bacteria can produce urease, which catalyzes the hydrolysis of urea to ammonia (NH3) and carbamate. A variety of urease inhibitors have been proposed to reduce NH3 volatilization by interfering with the urease activity. We report a quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) study on the mechanism employed for the inhibition of urease by three representative competitive inhibitors; namely, acetohydroxamic acid (AHA), hydroxyurea (HU), and N-(n-butyl)phosphorictriamide (NBPTO). The possible connections between the structural and thermodynamical properties and the experimentally observed inhibition efficiency were evaluated and characterized. We demonstrate that the binding affinity decreases in the order NBPTO >> AHA > HU in terms of the computed activation and reaction free energies. This trend also indicates that NBPTO shows the highest inhibitory activity and the lowest IC50 value of 2.1 nM, followed by AHA (42 μM) and HU (100 μM). It was also found that the X=O moiety (X = carbon or phosphorous) plays a crucial role in the inhibitor binding process. These findings not only elucidate why the potent urease inhibitors are effective but also have implications for the design of new inhibitors.

Investigation of Newly Synthesized Bis-Acyl-Thiourea Derivatives of 4-Nitrobenzene-1,2-Diamine for Their DNA Binding, Urease Inhibition, and Anti-Brain-Tumor Activities

Bis-acyl-thiourea derivatives, namely N,N'-(((4-nitro-1,2-phenylene)bis(azanediyl)) bis(carbonothioyl))bis(2,4-dichlorobenzamide) (UP-1), N,N'-(((4-nitro-1,2-phenylene) bis(azanediyl))bis(carbonothioyl))diheptanamide (UP-2), and N,N'-(((4-nitro-1,2-phenylene)bis(azanediyl))bis(carbonothioyl))dibutannamide (UP-3), were synthesized in two steps. The structural characterization of the derivatives was carried out by FTIR, 1H-NMR, and 13C-NMR, and then their DNA binding, anti-urease, and anticancer activities were explored. Both theoretical and experimental results, as obtained by density functional theory, molecular docking, UV-visible spectroscopy, fluorescence (Flu-)spectroscopy, cyclic voltammetry (CV), and viscometry, pointed towards compounds' interactions with DNA. However, the values of binding constant (Kb), binding site size (n), and negative Gibbs free energy change (ΔG) (as evaluated by docking, UV-vis, Flu-, and CV) indicated that all the derivatives exhibited binding interactions with the DNA in the order UP-3 > UP-2 > UP-1. The experimental findings from spectral and electrochemical analysis complemented each other and supported the theoretical analysis. The lower diffusion coefficient (Do) values, as obtained from CV responses of each compound after DNA addition at various scan rates, further confirmed the formation of a bulky compound-DNA complex that caused slow diffusion. The mixed binding mode of interaction as seen in docking was further verified by changes in DNA viscosity with varying compound concentrations. All compounds showed strong anti-urease activity, whereas UP-1 was found to have comparatively better inhibitory efficiency, with an IC50 value of 1.55 ± 0.0288 µM. The dose-dependent cytotoxicity of the synthesized derivatives against glioblastoma MG-U87 cells (a human brain cancer cell line) followed by HEK-293 cells (a normal human embryonic kidney cell line) indicated that UP-1 and UP-3 have greater cytotoxicity against both cancerous and healthy cell lines at 400 µM. However, dose-dependent responses of UP-2 showed cytotoxicity against cancerous cells, while it showed no cytotoxicity on the healthy cell line at a low concentration range of 40-120 µM.

Urease of Aspergillus fumigatus Is Required for Survival in Macrophages and Virulence.

The number of patients suffering from fungal diseases has constantly increased during the last decade. Among the fungal pathogens, the airborne filamentous fungus Aspergillus fumigatus can cause chronic and fatal invasive mold infections. So far, only three major classes of drugs (polyenes, azoles, and echinocandins) are available for the treatment of life-threatening fungal infections, and all present pharmacological drawbacks (e.g., low solubility or toxicity). Meanwhile, clinical antifungal-resistant isolates are continuously emerging. Therefore, there is a high demand for novel antifungal drugs, preferentially those that act on new targets. We studied urease and the accessory proteins in A. fumigatus to determine their biochemical roles and their influence on virulence. Urease is crucial for the growth on urea as the sole nitrogen source, and the transcript and protein levels are elevated on urea media. The urease deficient mutant displays attenuated virulence, and its spores are more susceptible to macrophage-mediated killing. We demonstrated that this observation is associated with an inability to prevent the acidification of the phagosome. Furthermore, we could show that a nickel-chelator inhibits growth on urea. The nickel chelator is also able to reverse the effects of urease on macrophage killing and phagosome acidification, thereby reducing virulence in systemic and trachea infection models. IMPORTANCE The development of antifungal drugs is an urgent task, but it has proven to be difficult due to many similarities between fungal and animal cells. Here, we characterized the urease system in A. fumigatus, which depends on nickel for activity. Notably, nickel is not a crucial element for humans. Therefore, we went further to explore the role of nickel-dependent urease in host-pathogen interactions. We were able to show that urease is important in preventing the acidification of the phagosome and therefore reduces the killing of conidia by macrophages. Furthermore, the deletion of urease shows reduced virulence in murine infection models. Taken together, we identified urease as an essential virulence factor of A. fumigatus. We were able to show that the application of the nickel-chelator dimethylglyoxime is effective in both in vitro and in vivo infection models. This suggests that nickel chelators or urease inhibitors are potential candidates for the development of novel antifungal drugs.