Laccases as Effective Tools in the Removal of Pharmaceutical Products from Aquatic Systems.

This review aims to provide a comprehensive overview of the application of bacterial and fungal laccases for the removal of pharmaceuticals from the environment. Laccases were evaluated for their efficacy in degrading pharmaceutical substances across various categories, including analgesics, antibiotics, antiepileptics, antirheumatic drugs, cytostatics, hormones, anxiolytics, and sympatholytics. The capability of laccases to degrade or biotransform these drugs was found to be dependent on their structural characteristics. The formation of di-, oligo- and polymers of the parent compound has been observed using the laccase mediator system (LMS), which is advantageous in terms of their removal via commonly used processes in wastewater treatment plants (WWTPs). Notably, certain pharmaceuticals such as tetracycline antibiotics or estrogen hormones exhibited degradation or even mineralization when subjected to laccase treatment. Employing enzyme pretreatment mitigated the toxic effects of degradation products compared to the parent drug. However, when utilizing the LMS, careful mediator selection is essential to prevent potential increases in environment toxicity. Laccases demonstrate efficiency in pharmaceutical removal within WWTPs, operating efficiently under WWTP conditions without necessitating isolation.

Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis.

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

Structure-Based Design and Pharmacophore-Based Virtual Screening of Combinatorial Library of Triclosan Analogues Active against Enoyl-Acyl Carrier Protein Reductase of Plasmodium falciparum with Favourable ADME Profiles.

Cost-effective therapy of neglected and tropical diseases such as malaria requires everlasting drug discovery efforts due to the rapidly emerging drug resistance of the plasmodium parasite. We have carried out computational design of new inhibitors of the enoyl-acyl carrier protein reductase (ENR) of Plasmodium falciparum (PfENR) using computer-aided combinatorial and pharmacophore-based molecular design. The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) complexation QSAR model was developed for triclosan-based inhibitors (TCL) and a significant correlation was established between the calculated relative Gibbs free energies of complex formation (∆∆Gcom) between PfENR and TCL and the observed inhibitory potencies of the enzyme (IC50exp) for a training set of 20 known TCL analogues. Validation of the predictive power of the MM-PBSA QSAR model was carried out with the generation of 3D QSAR pharmacophore (PH4). We obtained a reasonable correlation between the relative Gibbs free energy of complex formation ∆∆Gcom and IC50exp values, which explained approximately 95% of the PfENR inhibition data: pIC50exp=-0.0544×∆∆Gcom+6.9336,R2=0.95. A similar agreement was established for the PH4 pharmacophore model of the PfENR inhibition (pIC50exp=0.9754×pIC50pre+0.1596, R2=0.98). Analysis of enzyme-inhibitor binding site interactions suggested suitable building blocks to be used in a virtual combinatorial library of 33,480 TCL analogues. Structural information derived from the complexation model and the PH4 pharmacophore guided us through in silico screening of the virtual combinatorial library of TCL analogues to finally identify potential new TCL inhibitors effective at low nanomolar concentrations. Virtual screening of the library by PfENR-PH4 led to a predicted IC50pre value for the best inhibitor candidate as low as 1.9 nM. Finally, the stability of PfENR-TCLx complexes and the flexibility of the active conformation of the inhibitor for selected top-ranking TCL analogues were checked with the help of molecular dynamics. This computational study resulted in a set of proposed new potent inhibitors with predicted antimalarial effects and favourable pharmacokinetic profiles that act on a novel pharmacological target, PfENR.

Methodology for clinical research.

A clinical research requires a systematic approach with diligent planning, execution and sampling in order to obtain reliable and validated results, as well as an understanding of each research methodology is essential for researchers. Indeed, selecting an inappropriate study type, an error that cannot be corrected after the beginning of a study, results in flawed methodology. The results of clinical research studies enhance the repertoire of knowledge regarding a disease pathogenicity, an existing or newly discovered medication, surgical or diagnostic procedure or medical device. Medical research can be divided into primary and secondary research, where primary research involves conducting studies and collecting raw data, which is then analysed and evaluated in secondary research. The successful deployment of clinical research methodology depends upon several factors. These include the type of study, the objectives, the population, study design, methodology/techniques and the sampling and statistical procedures used. Among the different types of clinical studies, we can recognize descriptive or analytical studies, which can be further categorized in observational and experimental. Finally, also pre-clinical studies are of outmost importance, representing the steppingstone of clinical trials. It is therefore important to understand the types of method for clinical research. Thus, this review focused on various aspects of the methodology and describes the crucial steps of the conceptual and executive stages.

Ethical considerations regarding animal experimentation.

Animal experimentation is widely used around the world for the identification of the root causes of various diseases in humans and animals and for exploring treatment options. Among the several animal species, rats, mice and purpose-bred birds comprise almost 90% of the animals that are used for research purpose. However, growing awareness of the sentience of animals and their experience of pain and suffering has led to strong opposition to animal research among many scientists and the general public. In addition, the usefulness of extrapolating animal data to humans has been questioned. This has led to Ethical Committees' adoption of the 'four Rs' principles (Reduction, Refinement, Replacement and Responsibility) as a guide when making decisions regarding animal experimentation. Some of the essential considerations for humane animal experimentation are presented in this review along with the requirement for investigator training. Due to the ethical issues surrounding the use of animals in experimentation, their use is declining in those research areas where alternative in vitro or in silico methods are available. However, so far it has not been possible to dispense with experimental animals completely and further research is needed to provide a road map to robust alternatives before their use can be fully discontinued.

Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli.

Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active.

The production of laccases by white-rot fungi under solid-state fermentation conditions.

Laccases (E.C. 1.10.3.2) produced by white-rot fungi (WRF) can be widely used, but the high cost prevents their use in large-scale industrial processes. Finding a solution to the problem could involve laccase production by solid-state fermentation (SSF) simulating the natural growth conditions for WRF. SSF offers several advantages over conventional submerged fermentation (SmF), such as higher efficiency and productivity of the process and pollution reduction. The aim of this review is therefore to provide an overview of the current state of knowledge about the laccase production by WRF under SSF conditions. The focus is on variations in the up-stream process, fermentation and down-stream process and their impact on laccase activity. The variations of up-stream processing involve inoculum preparation, inoculation of the medium and formulation of the propagation and production media. According to the studies, the production process can be shortened to 5-7 days by the selection of a suitable combination of lignocellulosic material and laccase producer without the need for any additional components of the culture medium. Efficient laccase production was achieved by valorisation of wastes as agro-food, municipal wastes or waste generated from wood processing industries. This leads to a reduction of costs and an increase in competitiveness compared to other commonly used methods and/or procedures. There will be significant challenges and opportunities in the future, where SSF could become more efficient and bring the enzyme production to a higher level, especially in new biorefineries, bioreactors and biomolecular/genetic engineering.

The TLC-Bioautography as a Tool for Rapid Enzyme Inhibitors detection - A Review.

Microorganisms and plants can be important sources of many compounds with potential pharmaceutical applications. Extraction of these matrices is one of the ways of identifying the presence of inhibitory active substances against enzymes whose high activity leads to serious human diseases including cancer, Parkinson's or Crohn's diseases. The isolation and purification of inhibitors are time-consuming and expensive steps in the analysis of the crude extract and therefore, it is necessary to find a fast, efficient, and inexpensive method for screening extracts of interest. TLC-Bioautography combines the separation of the extract on a thin layer with its subsequent biological analysis. TLC-Bioautography methods have been developed for several classes of enzymes including oxidoreductases, hydrolases and isomerases, and there is a potential for developing functional methods for other classes of enzymes. This review summarizes known TLC-Bioautography methods and their applications for determining the presence of enzyme inhibitors in extracts and compares the effectiveness of different methodological approaches. It also indicates the current state and perspective of the development of TLC-Bioautography and its possible future applications.

Adhesion of fibroblast cells on thin films representing surfaces of polymeric scaffolds of human urethra rationalized by molecular models of integrin binding: cell adhesion on polymeric scaffolds for regenerative medicine.

This work combines experimental and computational study of Balb/3T3 clone A31 mouse embryo fibroblasts cell line adhesion and proliferation on fourteen different polymeric surfaces prepared from poly(dioxanone) (PDO), poly(glycolic acid) (PGA), poly(hydroxybutyrate) (PHB), and poly(L-lactic acid) (PLA), and their 1:1 mixtures. The study was done with the aim to explore the attractive interactions between various synthetic biomaterials and simple model of the cell attachment mechanism involving the trans-membrane protein integrin. The considered polymeric biodegradable biomaterials can be used as scaffolds for tissue engineering and regenerative urology. During the growth of new tissue, the polymer scaffold is replaced by the extracellular matrix (ECM) synthetized by the proliferating cells. The adhesion and proliferation experiments were done on thin polymer films produced by solvent casting. The computational approach used 3D molecular models of two layers of ordered parallel polymeric fibres, which formed quasi-planar nanosized models of the scaffold surface. Experimental data showed that PGA based polymer films promote the cell adhesion. Cell proliferation testing, performed by incubating the fibroblast cells with the studied polymer films, disclosed that PLA, PHB/PLA and PHB/PGA systems are able to support proliferation of Balb/3T3 clone A31 cells equal to the plain glass. Relative interaction energies between 3D models of polymeric films and the α2 I domain of the cell adhesion receptor integrin α2ß1 computed by molecular mechanics suggest that plain polymers PGA, PDO and mixtures PDO/PGA, PHB/PGA, and especially PGA/PLA display elevated affinity to the cell-attachment protein, which confirms the experimental observations. The combination of experimental and modelling approach can assist rational design of synthetic polymeric biomaterial for scaffolds of artificial human urethra that can be efficiently colonized by cells.

Ethics committees for clinical experimentation at international level with a focus on Italy.

Guiding legislation and associated bureaucracy for the ethical review of clinical trials observational studies and food related research play an important role in the competitiveness of a nation in the face of tough global competition to attract sponsors and investigators. This is of particular relevance in the case of multicentre trials and multidisciplinary research. Accordingly, in this report we tried to gather in-depth knowledge of the current role and practices of ethics committees nationwide in both clinical and research settings. This mini-review aims to describe the formulation and organization of ethical committees in Italy in order to provide a focus for deliberations on ethical issues in medical and scientific research in line with human rights, as set out in the European Union charter. Furthermore, we evaluated the impact of an institution's ethical committee intervention on reducing the time required to obtain an opinion from Research Ethics Committees by guiding investigators in addressing ethical issues in their proposed studies.

Purification of viral neuraminidase from inclusion bodies produced by recombinant Escherichia coli.

Neuraminidase (NA) is one of the targets for the development of new antivirals against the influenza virus. The recombinant Escherichia coli cells, namely the strains BL21(DE3)pLysS and ArcticExpress(DE3) were used to produce the influenza virus neuraminidase. Although the different conditions of induction were tested, the accumulation of over-expressed NA in insoluble fraction occurred independently of these conditions. The level of over-expressed protein represents 26.15 % of the total cellular proteins. Therefore, the aim of these study was to design the procedure for isolation of recombinant neuraminidase from IBs and subsequently its solubilization and refolding to its native and active form. The highest purity of IBs (86 %) was achieved after repeatedly washing for at least five times with 2 M urea. The best solubilizing agent for releasing NA from IBs was the solution of 8 M urea at pH 8.0 with 94.8 ± 0.4 mg/L released proteins. The most appropriate buffer for refolding of solubilized NA was found to be 50 mM Tris-HCl at pH 7.5 (102 ± 24.2 mg proteins) and the addition of glycerol or arginine had no stimulating effect on protein recovery. The determination of non-glycosylated activity of refolded NA monomer (Km = 0.51 g/L; Vmax = 9.73 U/mg; kcat = 8.76 s-1) using fetuin as a substrate in the coupled enzyme reaction system was the highlight of this work. This procedure provides a way to produce active form of NA monomer by recombinant E. coli cells.

Diversity of sialidases found in the human body - A review.

Sialidases are enzymes essential for numerous organisms including humans. Hydrolytic sialidases (EC 3.2.1.18), trans-sialidases and anhydrosialidases (intramolecular trans-sialidases, EC 4.2.2.15) are glycoside hydrolase enzymes that cleave the glycosidic linkage and release sialic acid residues from sialyl substrates. The paper summarizes diverse sialidases present in the human body and their potential impact on development of antiviral compounds - inhibitors of viral neuraminidases. It includes a brief overview of catalytic mechanisms of action of sialidases and describes the origin of sialidases in the human body. This is followed by description of the structure and function of sialidase families with a special focus on the GH33 and GH34 families. Various effects of sialidases on human body are also briefly described. Modulation of sialidase activity may be considered a useful tool for effective treatment of various diseases. In some cases, it is desired to completely suppress the activity of sialidases by suitable inhibitors. Specific sialidase inhibitors are useful for the treatment of influenza, epilepsy, Alzheimer's disease, diabetes, different types of cancer, or heart defects. Challenges and future directions are shortly depicted in the final part of the paper.

Antiviral agents against COVID-19: structure-based design of specific peptidomimetic inhibitors of SARS-CoV-2 main protease.

Despite the intense development of vaccines and antiviral therapeutics, no specific treatment of coronavirus disease 2019 (COVID-19), caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently available. Recently, X-ray crystallographic structures of a validated pharmacological target of SARS-CoV-2, the main protease (Mpro also called 3CLpro) in complex with peptide-like irreversible inhibitors have been published. We have carried out computer-aided structure-based design and optimization of peptidomimetic irreversible α-ketoamide Mpro inhibitors and their analogues using MM, MD and QM/MM methodology, with the goal to propose lead compounds with improved binding affinity to SARS-CoV-2 Mpro, enhanced specificity for pathogenic coronaviruses, decreased peptidic character, and favourable drug-like properties. The best inhibitor candidates designed in this work show largely improved interaction energies towards the Mpro and enhanced specificity due to 6 additional hydrogen bonds to the active site residues. The presented results on new SARS-CoV-2 Mpro inhibitors are expected to stimulate further research towards the development of specific anti-COVID-19 drugs.

Diagnostic and therapeutic implements based on advanced Biotechnology should be available in low-income countries.

N.A.

Structure-Based Design and in Silico Screening of Virtual Combinatorial Library of Benzamides Inhibiting 2-trans Enoyl-Acyl Carrier Protein Reductase of Mycobacterium tuberculosis with Favorable Predicted Pharmacokinetic Profiles.

BACKGROUND: During the previous decade a new class of benzamide-based inhibitors of 2-trans enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (Mt) with unusual binding mode have emerged. Here we report in silico design and evaluation of novel benzamide InhA-Mt inhibitors with favorable predicted pharmacokinetic profiles. METHODS: By using in situ modifications of the crystal structure of N-benzyl-4-((heteroaryl)methyl) benzamide (BHMB)-InhA complex (PDB entry 4QXM), 3D models of InhA-BHMBx complexes were prepared for a training set of 19 BHMBs with experimentally determined inhibitory potencies (half-maximal inhibitory concentrations IC50exp). In the search for active conformation of the BHMB1-19, linear QSAR model was prepared, which correlated computed gas phase enthalpies of formation (∆∆HMM) of InhA-BHMBx complexes with the IC50exp. Further, taking into account the solvent effect and entropy changes upon ligand, binding resulted in a superior QSAR model correlating computed complexation Gibbs free energies (∆∆Gcom). The successive pharmacophore model (PH4) generated from the active conformations of BHMBs served as a virtual screening tool of novel analogs included in a virtual combinatorial library (VCL) of compounds containing benzamide scaffolds. The VCL filtered by Lipinski's rule-of-five was screened by the PH4 model to identify new BHMB analogs. RESULTS: Gas phase QSAR model: -log10(IC50exp) = pIC50exp = -0.2465 × ∆∆HMM + 7.95503, R2 = 0.94; superior aqueous phase QSAR model: pIC50exp = -0.2370 × ∆∆Gcom + 7.8783, R2 = 0.97 and PH4 pharmacophore model: p IC 50 exp = 1.0013 × p IC 50 exp - 0.0085, R2 = 0.95. The VCL of more than 114 thousand BHMBs was filtered down to 73,565 analogs Lipinski's rule. The five-point PH4 screening retained 90 new and potent BHMBs with predicted inhibitory potencies IC50pre up to 65 times lower than that of BHMB1 (IC50exp = 20 nM). Predicted pharmacokinetic profile of the new analogs showed enhanced cell membrane permeability and high human oral absorption compared to current anti-tuberculotics. CONCLUSIONS: Combined use of QSAR models that considered binding of the BHMBs to InhA, pharmacophore model, and ADME properties helped to recognize bound active conformation of the benzamide inhibitors, permitted in silico screening of VCL of compounds sharing benzamide scaffold and identification of new analogs with predicted high inhibitory potencies and favorable pharmacokinetic profiles.

Virtual design of novel Plasmodium falciparum cysteine protease falcipain-2 hybrid lactone-chalcone and isatin-chalcone inhibitors probing the S2 active site pocket.

We report computer-aided design of new lactone-chalcone and isatin-chalcone (HLCIC) inhibitors of the falcipain-2 (PfFP-2). 3D models of 15 FP-2:HLCIC1-15 complexes with known observed activity (IC50exp) were prepared to establish a quantitative structure-activity (QSAR) model and linear correlation between relative Gibbs free energy of enzyme:inhibitor complex formation (ΔΔGcom) and IC50exp: pIC50exp = -0.0236 × ΔΔGcom+5.082(#); R2 = 0.93. A 3D pharmacophore model (PH4) derived from the QSAR directed our effort to design novel HLCIC analogues. During the design, an initial virtual library of 2621440 HLCIC was focused down to 18288 drug-like compounds and finally, PH4 screened to identify 81 promising compounds. Thirty-three others were added from an intuitive substitution approach intended to fill better the enzyme S2 pocket. One hundred and fourteen theoretical IC50 (IC50pre) values were predicted by means of (#) and their pharmacokinetics (ADME) profiles. More than 30 putative HLCICs display IC50pre 100 times superior to that of the published most active training set inhibitor HLCIC1.

Current state and prospects of biotechnology in Central and Eastern European countries. Part I: Visegrad countries (CZ, H, PL, SK).

Innovation is a key determinant of sustainable growth. Biotechnology (BT) is one such industry that has witnessed a revolution in innovative ideas leading to the founding of many new companies based on providing products, solutions and services, stretching from the food industry to environmental remediation, and new medicines. BT holds much promise for the development of national and local economies, however, this requires a strategic approach involving actors within government, industry, and academia working in concert to maximize this potential. This first article reviews the current "state of play" in the field of BT within the Central Eastern European (CEE) countries. For the purposes of this article, CEE refers to the countries of Czech Republic, Hungary, Poland, and Slovakia (the so-called Visegrad - V4 countries). We examine the components that support the creation and development of a BT sector in CEE and also highlight the barriers to these objectives. Clearly setting priorities for the countries' policy agenda, as well as the alignment of Smart Specialization Strategy will help to focus efforts. Recent investments in R&D infrastructure within CEE have been substantial, but conditions will need to be optimized to harness these largely European investments for effective use towards SME high-tech development.

Current state and prospects of biotechnology in Central and Eastern European countries. Part II: new and preaccession EU countries(CRO, RO, B&H, SRB).

Innovation holds the potential for economic prosperity. Biotechnology (BT) has proved to be a viable vehicle for the development and utilization of technologies, which has brought not only advances to society, but also career opportunities to nation-states that have enabling conditions. In this review, we assess the current state of BT-related activities within selected new and preaccession EU countries (NPA) of CEE region namely Croatia, Romania, Bosnia and Herzegovina and Serbia, examining educational programs, research activity, enterprises, and the financing systems. The field of BT covers a broad area of activities, including medical, food and agriculture, aquaculture or marine, environmental, biofuels, bioinformatics, and many others. Under the European Commission (EC), member-states are to set their Research and Innovation Strategies for Smart Specialization (RIS3), to identify priorities or strengths in order to develop knowledge intensive economies. As the four countries highlighted in this review are in the early stages of implementing RIS3 or have not yet fully formulated, it presents an opportunity to learn from the successes and failures of those that have already received major structural funds from the EC. A critical point will be the ability of the public and private sectors' actors to align, in the implementation of RIS3 as new investment instruments emerge, and to concentrate efforts on a few select target goals, rather than distribute funding widely without respect to a long-term vision.

Methods and Current Trends in Determination of Neuraminidase Activity and Evaluation of Neuraminidase Inhibitors.

This review focuses on the methods and current trends in determination of neuraminidases (NAs) activity and evaluation of neuraminidase inhibitors (NAIs) by means of biochemical assays. These methods can be used, in principle, for any type of sialidase, with regard to substrate specificity and optimal conditions for enzymatic reaction. Considering the range of organisms producing sialidases, this review omits cell-based assays (plaque assays and study of cytopathic effect) and animal model studies, which are reviewed elsewhere concerning specific organisms. The present review also provides the brief introductory survey of role of sialic acids and neuraminidases, but main focus is on the methods of determining neuraminidase activity and evaluating neuraminidase inhibitors. The future prospect of improvement in analytical techniques regarding the enzymatic activity of sialidases is briefly outlined.