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1.
Sci Rep ; 14(1): 3769, 2024 02 14.
Article in English | MEDLINE | ID: mdl-38355917

ABSTRACT

The current study provides field experimental data that support the use of γ-polyglutamic acid (γ-PGA) in drought stress and proposes its application in grassland management. We hypothesized that water treatment combined with PGA application to sandy soil would reduce drought stress in grasslands more effectively than watering alone. A randomized block design was used, with three replicate watering blocks (no watering, weekly watering, and monthly watering) and PGA treatments at four different concentrations (0%, 0.3%, 1%, and 2% PGA). The results showed that PGA acts as a biostimulant, alleviating the effects of stress in plants by: (1) increasing the availability of ions, especially K+, Zn2+, Mn2+, Fe2+/3+, Ca2+, and Mg2+, as well as N-NH4+, and N-NO3-, (2) elongating plant roots, (3) increasing the aboveground biomass, (4) improving the resprouting capacity of the dominant grass Nardus stricta, and (5) improving the regeneration of dicotyledons. In the case of meadows on sandy soils, the use of low PGA concentrations (0.3% or 1%) was the most beneficial for the availability of macro- and microelements and improving the functional traits of plants. Irrigation had a greater effect than using PGA only for the dicotyledon to monocotyledon ratio.


Subject(s)
Magnoliopsida , Polyglutamic Acid/analogs & derivatives , Soil , Grassland , Sand , Droughts , Plants , Poaceae
2.
J Chem Inf Model ; 63(12): 3669-3687, 2023 06 26.
Article in English | MEDLINE | ID: mdl-37285179

ABSTRACT

Toll-like receptors (TLRs) are transmembrane proteins that recognize various molecular patterns and activate signaling that triggers the immune response. In this review, our goal is to summarize how, in recent years, various computational solutions have contributed to a better understanding of TLRs, regarding both their function and mechanism of action. We update the recent information about small-molecule modulators and expanded the topic toward next-generation vaccine design, as well as studies of the dynamic nature of TLRs. Also, we underline problems that remain unsolved.


Subject(s)
Signal Transduction , Toll-Like Receptors , Toll-Like Receptors/metabolism
3.
PLoS One ; 18(2): e0280776, 2023.
Article in English | MEDLINE | ID: mdl-36827335

ABSTRACT

The haloalkane dehalogenase LinB is a well-known enzyme that contains buried active site and is used for many modelling studies. Using classical molecular dynamics simulations of enzymes and substrates, we searched for transient binding sites on the surface of the LinB protein by calculating maps of enzyme-ligand interactions that were then transformed into sparse matrices. All residues considered as functionally important for enzyme performance (e.g., tunnel entrances) were excluded from the analysis to concentrate rather on non-obvious surface residues. From a set of 130 surface residues, twenty-six were proposed as a promising improvement of enzyme performance. Eventually, based on rational selection and filtering out the potentially unstable mutants, a small library of ten mutants was proposed to validate the possibility of fine-tuning the LinB protein. Nearly half of the predicted mutant structures showed improved activity towards the selected substrates, which demonstrates that the proposed approach could be applied to identify non-obvious yet beneficial mutations for enzyme performance especially when obvious locations have already been explored.


Subject(s)
Hydrolases , Molecular Dynamics Simulation , Binding Sites , Hydrolases/metabolism , Catalytic Domain
4.
J Chem Inf Model ; 62(24): 6803-6811, 2022 12 26.
Article in English | MEDLINE | ID: mdl-36374085

ABSTRACT

Different methods for tunnel identification, geometry-based and small-molecule tracking approaches, were compared to provide their benefits and pitfalls. Results obtained for both crystal structures and molecular dynamics (MD) simulations were analyzed to investigate if a more computationally demanding method would be beneficial. Careful examination of the results is essential for the low-diameter tunnel description, and assessment of the tunnel functionality based only on their geometrical parameters is challenging. We showed that the small-molecule tracking approach can provide a detailed description of the system; however, it can also be the most computationally demanding.


Subject(s)
Molecular Dynamics Simulation
6.
PLoS Comput Biol ; 18(5): e1010119, 2022 05.
Article in English | MEDLINE | ID: mdl-35580137

ABSTRACT

The evolutionary variability of a protein's residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to α/ß-hydrolase fold proteins-one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels' evolution analysis based on entropy values calculated for tunnels' residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a 'perforation' mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzyme's performance and can be applied widely for enzymes with buried active sites.


Subject(s)
Epoxide Hydrolases , Hydrolases , Binding Sites , Catalytic Domain , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/genetics , Epoxide Hydrolases/metabolism , Hydrolases/chemistry , Hydrolases/metabolism , Proteins
7.
Biomolecules ; 12(3)2022 03 04.
Article in English | MEDLINE | ID: mdl-35327592

ABSTRACT

Congenital Disorders of Glycosylation (CDG) are multisystemic metabolic disorders showing highly heterogeneous clinical presentation, molecular etiology, and laboratory results. Here, we present different transferrin isoform patterns (obtained by isoelectric focusing) from three female patients harboring the ALG13 c.320A>G mutation. Contrary to other known variants of type I CDGs, where transferrin isoelectric focusing revealed notably increased asialo- and disialotransferrin fractions, a normal glycosylation pattern was observed in the probands. To verify this data and give novel insight into this variant, we modeled the human Alg13 protein and analyzed the dynamics of the apo structure and the complex with the UDP-GlcNAc substrate. We also modeled the Alg13-Alg14 heterodimer and ran multiple simulations of the complex in the presence of the substrate. Finally, we proposed a plausible complex formation mechanism.


Subject(s)
Congenital Disorders of Glycosylation , Biological Variation, Population , Congenital Disorders of Glycosylation/genetics , Female , Glycosylation , Humans , Mutation , N-Acetylglucosaminyltransferases
8.
Comput Struct Biotechnol J ; 20: 193-205, 2022.
Article in English | MEDLINE | ID: mdl-35024092

ABSTRACT

Enzymes with buried active sites maintain their catalytic function via a single tunnel or tunnel network. In this study we analyzed the functionality of soluble epoxide hydrolases (sEHs) tunnel network, by comparing the overall enzyme structure with the tunnel's shape and size. sEHs were divided into three groups based on their structure and the tunnel usage. The obtained results were compared with known substrate preferences of the studied enzymes, as well as reported in our other work evolutionary analyses data. The tunnel network architecture corresponded well with the evolutionary lineage of the source organism and large differences between enzymes were observed from long fragments insertions. This strategy can be used during protein re-engineering process for large changes introduction, whereas tunnel modification can be applied for fine-tuning of enzyme.

9.
PLoS One ; 17(1): e0262482, 2022.
Article in English | MEDLINE | ID: mdl-35015795

ABSTRACT

Based on previous large-scale in silico screening several factor Xa inhibitors were proposed to potentially inhibit SARS-CoV-2 Mpro. In addition to their known anticoagulants activity this potential inhibition could have an additional therapeutic effect on patients with COVID-19 disease. In this study we examined the binding of the Apixaban, Betrixaban and Rivaroxaban to the SARS-CoV-2 Mpro with the use of the MicroScale Thermophoresis technique. Our results indicate that the experimentally measured binding affinity is weak and the therapeutic effect due to the SARS-CoV-2 Mpro inhibition is rather negligible.


Subject(s)
Coronavirus M Proteins/antagonists & inhibitors , Factor Xa Inhibitors/chemistry , SARS-CoV-2/metabolism , Benzamides/chemistry , Benzamides/metabolism , Binding Sites , COVID-19/virology , Coronavirus M Proteins/metabolism , Factor Xa Inhibitors/metabolism , Humans , Molecular Dynamics Simulation , Protein Binding , Protein Stability , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyridines/chemistry , Pyridines/metabolism , Pyridones/chemistry , Pyridones/metabolism , Rivaroxaban/chemistry , Rivaroxaban/metabolism , SARS-CoV-2/isolation & purification , COVID-19 Drug Treatment
10.
Pharmaceuticals (Basel) ; 14(11)2021 Nov 12.
Article in English | MEDLINE | ID: mdl-34832935

ABSTRACT

In the present study we tested, using the microscale thermophoresis technique, a small library of thionocarbamates, thiolocarbamates, sulfide and disulfide as potential lead compounds for SARS-CoV-2 Mpro drug design. The successfully identified binder is a representative of the thionocarbamates group with a high potential for future modifications aiming for higher affinity and solubility. The experimental analysis was extended by computational studies that show insufficient accuracy of the simplest and widely applied approaches and underline the necessity of applying more advanced methods to properly evaluate the affinity of potential SARS-CoV-2 Mpro binders.

11.
Drug Discov Today ; 26(8): 1914-1921, 2021 08.
Article in English | MEDLINE | ID: mdl-34082135

ABSTRACT

Human soluble epoxide hydrolase (hsEH) is involved in the hydrolysis of epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory properties. Given that EET conversion generates nonbioactive molecules, inhibition of this enzyme would be beneficial. Past decades of work on hsEH inhibitors resulted in numerous potential compounds, of which a hundred hsEH-ligand complexes were crystallized and deposited in the Protein Data Bank (PDB). We analyzed all deposited hsEH-ligand complexes to gain insight into the binding of inhibitors and to provide feedback on the future drug design processes. We also reviewed computationally driven strategies that were used to propose novel hsEH inhibitors.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Drug Design , Epoxide Hydrolases/antagonists & inhibitors , Eicosanoids/metabolism , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/metabolism , Humans , Ligands
12.
Int J Mol Sci ; 22(4)2021 Feb 19.
Article in English | MEDLINE | ID: mdl-33669738

ABSTRACT

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious global health threat. Since no specific therapeutics are available, researchers around the world screened compounds to inhibit various molecular targets of SARS-CoV-2 including its main protease (Mpro) essential for viral replication. Due to the high urgency of these discovery efforts, off-target binding, which is one of the major reasons for drug-induced toxicity and safety-related drug attrition, was neglected. Here, we used molecular docking, toxicity profiling, and multiple molecular dynamics (MD) protocols to assess the selectivity of 33 reported non-covalent inhibitors of SARS-CoV-2 Mpro against eight proteases and 16 anti-targets. The panel of proteases included SARS-CoV Mpro, cathepsin G, caspase-3, ubiquitin carboxy-terminal hydrolase L1 (UCHL1), thrombin, factor Xa, chymase, and prostasin. Several of the assessed compounds presented considerable off-target binding towards the panel of proteases, as well as the selected anti-targets. Our results further suggest a high risk of off-target binding to chymase and cathepsin G. Thus, in future discovery projects, experimental selectivity assessment should be directed toward these proteases. A systematic selectivity assessment of SARS-CoV-2 Mpro inhibitors, as we report it, was not previously conducted.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , COVID-19/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Drug Discovery/methods , Humans , Molecular Docking Simulation/methods , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , SARS-CoV-2/enzymology
13.
Diagnostics (Basel) ; 10(10)2020 10 14.
Article in English | MEDLINE | ID: mdl-33066491

ABSTRACT

(1) Background: Molybdenum cofactor deficiency type B (MOCODB, #252160) is a rare autosomal recessive metabolic disorder characterized by intractable seizures of neonatal-onset, muscular spasticity, accompanying with hypouricemia, elevated urinary sulfite levels and craniofacial dysmorphism. Thirty-five patients were reported to date. (2) Methods: Our paper aimed to delineate the disease genotype by presenting another patient, in whom a novel, in-frame variant within the MOCS2 gene was identified. (3) Results: Exome sequencing led to the identification of a novel variant in the MOCS2 gene-c.472_477del of unknown significance (VUS). (4) Conclusions: To prove the clinical significance of the mentioned variant, analysis of the possible mutation consequences on molecular level with the use of the available crystal structure of the human molybdopterin synthase complex was of great importance. Moreover, a potential pathomechanism resulting from a molecular defect was presented, giving original insight into the current knowledge on this rare disease, including treatment options.

14.
Int J Mol Sci ; 21(9)2020 Apr 28.
Article in English | MEDLINE | ID: mdl-32353978

ABSTRACT

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mixed-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar severe acute respiratory syndrome (SARS) Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins showed major differences in both shape and size, indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site's conformational changes during the simulation time indicated its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicated that the virus' mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.


Subject(s)
Betacoronavirus/enzymology , Cysteine Endopeptidases/chemistry , Drug Design , Protease Inhibitors/pharmacology , Small Molecule Libraries , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/genetics , Binding Sites , COVID-19 , Catalytic Domain , Coronavirus 3C Proteases , Coronavirus Infections , Crystallography, X-Ray , Cysteine Endopeptidases/genetics , Drug Evaluation, Preclinical , Evolution, Molecular , Models, Molecular , Molecular Dynamics Simulation , Mutation , Pandemics , Pneumonia, Viral , Severe acute respiratory syndrome-related coronavirus/enzymology , SARS-CoV-2 , Solvents , Thermodynamics , Viral Nonstructural Proteins/genetics
15.
Int J Mol Sci ; 21(7)2020 Mar 26.
Article in English | MEDLINE | ID: mdl-32225102

ABSTRACT

Loops are the most variable and unorganized elements of the secondary structure of proteins. Their ability to shift their shape can play a role in the binding of small ligands, enzymatic catalysis, or protein-protein interactions. Due to the loop flexibility, the positions of their residues in solved structures show the largest B-factors, or in a worst-case scenario can be unknown. Based on the loops' movements' timeline, they can be divided into slow (static) and fast (flexible). Although most of the loops that are missing in experimental structures belong to the flexible loops group, the computational tools for loop reconstruction use a set of static loop conformations to predict the missing part of the structure and evaluate the model. We believe that these two loop types can adopt different conformations and that using scoring functions appropriate for static loops is not sufficient for flexible loops. We showed that common model evaluation methods, are insufficient in the case of flexible solvent-exposed loops. Instead, we recommend using the potential energy to evaluate such loop models. We provide a novel model selection method based on a set of geometrical parameters to distinguish between flexible and static loops without the use of molecular dynamics simulations. We have also pointed out the importance of water network and interactions with the solvent for the flexible loop modeling.


Subject(s)
Molecular Dynamics Simulation , Sequence Analysis, Protein/methods , Animals , Aspergillus niger/enzymology , Bombyx/enzymology , Epoxide Hydrolases/chemistry , Fungal Proteins/chemistry , Insect Proteins/chemistry , Protein Conformation, alpha-Helical , Protein Domains
16.
Comput Struct Biotechnol J ; 18: 355-365, 2020.
Article in English | MEDLINE | ID: mdl-32123557

ABSTRACT

Water molecules maintain proteins' structures, functions, stabilities and dynamics. They can occupy certain positions or pass quickly via a protein's interior. Regardless of their behaviour, water molecules can be used for the analysis of proteins' structural features and biochemical properties. Here, we present a list of several software programs that use the information provided by water molecules to: i) analyse protein structures and provide the optimal positions of water molecules for protein hydration, ii) identify high-occupancy water sites in order to analyse ligand binding modes, and iii) detect and describe tunnels and cavities. The analysis of water molecules' distribution and trajectories sheds a light on proteins' interactions with small molecules, on the dynamics of tunnels and cavities, on protein composition and also on the functionality, transportation network and location of functionally relevant residues. Finally, the correct placement of water molecules in protein crystal structures can significantly improve the reliability of molecular dynamics simulations.

17.
J Pharm Biomed Anal ; 185: 113216, 2020 Jun 05.
Article in English | MEDLINE | ID: mdl-32155543

ABSTRACT

The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone. Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium. The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram'-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample. In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative's biological properties.


Subject(s)
Anticarcinogenic Agents/pharmacokinetics , Genistein/pharmacokinetics , Neoplasms/drug therapy , Administration, Oral , Animals , Anticarcinogenic Agents/administration & dosage , Anticarcinogenic Agents/chemistry , Biological Availability , Caco-2 Cells , Cell Membrane Permeability , Female , Genistein/administration & dosage , Genistein/analogs & derivatives , Genistein/chemistry , Humans , Intestinal Absorption , Models, Animal , Molecular Structure , Permeability , Rats , Structure-Activity Relationship
18.
Bioinformatics ; 36(8): 2599-2601, 2020 04 15.
Article in English | MEDLINE | ID: mdl-31860077

ABSTRACT

MOTIVATION: Tunnels, pores, channels, pockets and cavities contribute to proteins architecture and performance. However, analysis and characteristics of transportation pathways and internal binding cavities are performed separately. We aimed to provide universal tool for analysis of proteins integral interior with access to detailed information on the ligands transportation phenomena and binding preferences. RESULTS: AQUA-DUCT version 1.0 is a comprehensive method for macromolecules analysis from the intramolecular voids perspective using small ligands as molecular probes. This version gives insight into several properties of macromolecules and facilitates protein engineering and drug design by the combination of the tracking and local mapping approach to small ligands. AVAILABILITY AND IMPLEMENTATION: http://www.aquaduct.pl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
Proteins , Software , Ligands , Macromolecular Substances , Protein Engineering
19.
Biomolecules ; 9(6)2019 05 31.
Article in English | MEDLINE | ID: mdl-31159273

ABSTRACT

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.


Subject(s)
Glucose-6-Phosphate Isomerase/genetics , Glucose-6-Phosphate Isomerase/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Pyrococcus furiosus/enzymology , Temperature , Enzyme Inhibitors/pharmacology , Glucose-6-Phosphate Isomerase/antagonists & inhibitors , Glucose-6-Phosphate Isomerase/chemistry , Manganese/metabolism , Molecular Dynamics Simulation , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/chemistry , Protein Conformation , Protein Engineering , Water/metabolism
20.
Biomolecules ; 8(4)2018 11 12.
Article in English | MEDLINE | ID: mdl-30424576

ABSTRACT

Several different approaches are used to describe the role of protein compartments and residues in catalysis and to identify key residues suitable for the modification of the activity or selectivity of the desired enzyme. In our research, we applied a combination of molecular dynamics simulations and a water tracking approach to describe the water accessible volume of Solanum tuberosum epoxide hydrolase. Using water as a molecular probe, we were able to identify small cavities linked with the active site: (i) one made up of conserved amino acids and indispensable for the proper positioning of catalytic water and (ii) two others in which modification can potentially contribute to enzyme selectivity and activity. Additionally, we identified regions suitable for de novo tunnel design that could also modify the catalytic properties of the enzyme. The identified hot-spots extend the list of the previously targeted residues used for modification of the regioselectivity of the enzyme. Finally, we have provided an example of a simple and elegant process for the detailed description of the network of cavities and tunnels, which can be used in the planning of enzyme modifications and can be easily adapted to the study of any other protein.


Subject(s)
Epoxide Hydrolases/chemistry , Solanum tuberosum/enzymology , Water/chemistry , Amino Acids/chemistry , Evolution, Molecular , Molecular Dynamics Simulation
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