Viral proteinases--possible targets of antiviral drugs.

Viral infections represent various types of human, veterinary and plant diseases with a significant economic, ethic and demographic impact. Over the years a significant effort has been made to develop various means of prevention and therapy of viral diseases. Proteinases play an important role in the process of virus replication as well as in the pathophysiology of many viral diseases. The aim of this review is to assess the prospects of the application of proteinase inhibitors in antiviral therapy and to characterize viral proteinases of various classes. Six Human immunodeficiency virus (HIV) proteinase inhibitors have been approved for therapeutic use and can serve as examples of prospective application ofproteinase inhibitors to antiviral therapy.

A kinetic distribution model of evaporation, biosorption and biodegradation of polychlorinated biphenyls (PCBs) in the suspension of Pseudomonas stutzeri.

Kinetics of distribution of PCBs in an active bacterial suspension of Pseudomonas stutzeri was studied by monitoring the evaporated amounts and the concentration remaining in the liquid medium with the biomass. To determine the biodegradation rate constants of the individual congeners of the PCB formulation Delor 103, a model considering biosorption, evaporation, and primary biodegradation constructed previously was used. Rate constants of biodegradation imply that biodegradation of individual congeners is structure-dependent process. Biodegradability decreases with increasing number of chlorine substituents in the molecule, especially if they are in the ortho and para positions. On the other hand, the increasing number of free ortho and meta positions in the biphenyl molecule leads to better biodegradability. For a simple empirical determination of the influence of the chlorine substitution pattern on biodegradability, the di- and trichlorobiphenyl rate constants of biodegradation were analysed.

Binding modes of PCBs to a degrading enzyme: a receptor-mapping study.

The binding site of a PCB-degrading enzyme was mapped using the published data on biodegradation rates of individual PCB congeners by the Acinetobacter P6 strain. For this purpose an approach allowing for multiple binding modes of individual congeners, resulting from the symmetry of the biphenyl skeleton, was used. The effect of substitution patterns and conformational flexibility of individual congeners on their binding to a protein were investigated. The resulting map of the binding site is described by three parameters that indicate the importance of positions 4, 5', 5, 2' in a basic substitution pattern, the first two being favourable while the other two unfavourable for binding. An incorporation of conformational energy dependences of individual ligands into the model showed that ligand's conformation is either not a limiting factor for binding or that ligands bind in their relaxed conformations.

Influence of new fenpropimorph fungicides on the growth and sterol composition in Saccharomyces cerevisiae: relationship between structure and activity.

The toxicity of fenpropimorph and seven newly synthesized analogues against Saccharomyces cerevisiae has been determined in liquid media. The inhibitory effect of the most efficient derivative is 120 times more than that of standard fenpropimorph. The non-linear relationship between hydrophobicity and toxicity indicates that the binding of the compounds to the receptors does not differ and so the differences in toxicity reflect changes in the rate of metabolism. The presence of inhibitors in the fermentation medium resulted in a reduction in harvested biomass and lipid yield, and changes in sterol composition -- the amount of ergosterol decreased whereas the amounts of lanosterol, dihydroergosterol and squalene increased. The toxicity of the compounds was most influenced by their lipophilicity. Use of this information could lead to development of more potent ergosterol inhibitors.

Kinetics of subcellular distribution of compounds in simple biosystems and its use in QSAR.

An explicit expression describing the kinetics of distribution and, in some cases, biological activity in drugs and other anthropogenic chemicals in a four-compartment system consisting of a catenary chain of alternating aqueous and lipoid phases is derived. Substitution of the transport rate parameters by their appropriate relations to the reference partition coefficient converts the kinetic equations into the quantitative structure-time-activity relationship (QSTAR) or its fixed-time equivalent, QSAR. The resulting expression describes satisfactorily the published data on antibacterial activity of n-alkyl amines as a function of their hydrophobicity. The hydrophobicity-activity profile consists of two or three smoothly connected linear parts. The model can be used in drug design, pharmacokinetics, and toxicology for description of the distribution of compounds in simple biosystems and in environmental sciences to predict the fate and effects of anthropogenic chemicals in ecological systems.

Relationships between the structure, cytotoxicity and hydrophobicity of quinazoline derivatives by quantitative structure-activity relationship.

Cytotoxicities of 93 quinazoline derivatives against HeLa cells have been determined as the isoeffective concentrations inhibiting, after a single dose, the protein synthesis to 50% of the control amount after 48 h incubation. The dependence of cytotoxicity on hydrophobicity of the studied derivatives has been described using a previously published model-based approach. The studied derivatives are classified into nine classes each forming a smooth hydrophobicity-cytotoxicity curve. Owing to the acceptable agreement between the model and the data it can be inferred that: (1) the compounds except two derivatives bind to the receptors with approximately the same affinity; (2) the criterion for the classification is the different rate of metabolism. The results represent a basis for a rational development of more potent quinazoline derivatives.

Model-based QSAR for ionizable compounds: toxicity of phenols against Tetrahymena pyriformis.

The previously described pseudo-equilibrium model for kinetics of distribution of extraneous chemicals in biological systems has been used for the development of the QSAR equation expressing toxicity of phenols against Tetrahymena pyriformis as a non-linear function of their hydrophobicity and acidity. The model assumes binding of all the tested compounds to a single class of the receptors (i.e., it does not discriminate between possible narcotic and respiratory uncoupling modes of action of phenols) with the association constant related to the electronic structure of the molecules rather than to their hydrophobicity. Non-linear regression analysis revealed satisfying agreement between the model and published biological data measured after a single dose.

Effect of chlorobenzoates on the degradation of polychlorinated biphenyls (PCB) by Pseudomonas stutzeri.

Chlorobenzoic acids (CBA) are frequently dead-end products of partial aerobic biodegradation of polychlorinated biphenyls (PCB). When CBA produced from PCB accumulate in the growth medium, they can inhibit the bacterial growth and consequently, slow down PCB biodegradation. In this study, the effects of seven mono- and dichlorinated CBA on growth of Pseudomonas stutzeri on different substrates and on the PCB degradation by this strain in a liquid mineral medium were tested. 3-CBA was the strongest growth inhibitor for P. stutzeri growing on glucose, benzoate and biphenyl. It was found to inhibit heavily the elimination of some di- and trichlorinated biphenyls. In contrast, its influence on the elimination of more chlorinated congeners was much less significant.

Degradation of PCB in different soils by inoculated Alcaligenes xylosoxidans.

The degradation of PCB in soils by the biphenyl-utilising strain Alcaligenes xylosoxidans was studied in different soil types. In addition to the congener specificity, significant differences in the degradation of PCB by the strain in the different soil types were observed. Efficiency of degradation was generally better in sterilised soils, but the differences were not as significant as the differences observed between different soil types. These results indicate that the degradation of PCB is probably related not only to the capabilities of the strain employed and quality and amount of competitive species inhabiting the soils, but also to the soil sorption of the PCB congeners. Degradation is faster in the soils containing an intermediate amount of organic carbon with a high portion of total and aromatic carbon in humic acids.

Functionally essential, invariant glutamate near the C-terminus of strand beta 5 in various (alpha/beta)8-barrel enzymes as a possible indicator of their evolutionary relatedness.

Twelve different (alpha/beta)8-barrel enzymes belonging to three structurally distinct families were found to contain, near the C-terminus of their strand beta 5, a conserved invariant glutamic acid residue that plays an important functional role in each of these enzymes. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif owing to their mutual evolutionary relatedness. For this purpose, the sequence region around the well conserved fifth beta-strand of alpha-amylase containing catalytic glutamate (Glu230, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The isolated sequence stretches of the 12 (alpha/beta)8-barrels are discussed from both the sequence-structural and the evolutionary point of view, the invariant glutamate residue being proposed to be a joining feature of the studied group of enzymes remaining from their ancestral (alpha/beta)8-barrel.

Model-based description of distribution of chemicals in biosystems for the continuous dose.

The distribution of xenobiotics in a morphologically compartmentalised biosystem is described for the continuous dose of readily metabolised chemicals. The time hierarchy of individual processes the compounds undergo in the biosystem (transport-considered as uni-directional due to metabolism, protein binding, and metabolism) is used to simplify the model description. The explicit expressions for the steady-state concentrations (ci) in individual compartments as a function of transport rate constants and metabolism rate constants are derived. Further substantial simplification can be achieved if the phases of the test biosystem can be considered as approximately identical. Under the conditions ci can be used to calculate the explicit expressions for the average concentrations in all aqueous phases and in all membranes. The model parameters (the membrane/water partition coefficient, the water-to-membrane transport rate constant) are substituted by the reference (usually 1-octanol/water) partition coefficient using plausible mutual relationships. The dependences of the steady-state concentrations in individual compartments as well as the average quantities in all aqueous phases and in all membranes on hydrophobicity and on the metabolism rate constant are analysed in detail. The presented explicit expressions for the steady-state concentrations create a firm basis for: (1) derivation of model-based QSAR equations for toxicity caused by individual mechanisms of action and for further quantities of environmental interest like bioconcentration factors and total metabolism rates as well as for (2) scaling and comparison of QSAR equations among individual organisms.

Assessment of QSARS for Predicting Fate and Effects of Chemicals in the Environment: An International European Project.

Abstract In 1993, an international project on QSAR has been started with funding from the Commission of the European Union. The first part of the project is focused on preparing an overview of existing models for the prediction of environmental parameters such as bioconcentration, sorption, degradation and ecotoxicity. Emphasis will be given to defining the limitations of the models. Since all models, including QSARs, have their limitations, it is important that these limitations are known in case QSARs are actually used and applied within the risk assessment context. The second part of the project is directed towards experimental research on new developments with emphasis on the use of multivariate techniques and quantum chemical properties. In this short paper, a general outline of the project will be given, as well as some first results. Results of experimental work within this project will be published in the proceedings of the 6th International Workshop on QSAR in Environmental Sciences and will appear in this same journal.

Evolution of parallel beta/alpha-barrel enzyme family lightened by structural data on starch-processing enzymes.

The parallel beta/alpha-barrel domain consisting of eight parallel beta-sheets surrounded by eight alpha-helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day beta/alpha-barrels, a challenging question remains unanswered: How has the parallel beta/alpha-barrel fold arisen? Although the complete sequence comparison of all beta/alpha-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of alpha-amylase as structural templates. Since many starch-processing enzymes adopt the parallel beta/alpha-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded beta/alpha-barrel enzyme family.

Relationship between antifungal activity and hydrophobicity of kojic acid derivatives.

Twenty three kojic acid derivatives were tested in the agar diffusion test against twenty five dermatophytic fungi. The inhibitory effects at equal doses (10 mg/L) were correlated with the 1-octanol-water partition coefficients of the compounds. An improvement in antifungal activity can be achieved by increasing the hydrophobicity of the compounds.

Fermentation of starch to ethanol by a co-culture of Saccharomycopsis fibuligera and Saccharomyces cerevisiae.

Static fermentation of starch to ethanol by a co-culture of Saccharomycopsis fibuligera and Saccharomyces cerevisiae without addition of nutritional supplements was investigated with respect to initial starch concentration, pH of the media and initial dry weight ratio of Sps. fibuligera to Sacc. cerevisiae biomass (I R).Optimal conditions for ethanol production were: starch from 20 to 30 g/l; initial pH values from 5.8 to 6.0; and I R values of 2.0 or 3.0. The highest attained ethanol concentration, 13.7 g/l, represented 88% of the theoretical yield.

Degradation of Delor 103, a technical mixture of polychlorinated biphenyls, by selected bacteria.

Ability to utilize a technical mixture of polychlorinated biphenyls (PCB), Delor 103, as the sole carbon source, has been tested in 14 bacterial strains. For the five best growing strains (Alcaligenes latus, Alcalgenes eutrophus, Comamonas testosteroni, Micrococcus varians and Pseudomonas putida), the dependence of the degradation of individual PCB congeners on the number of chlorine substituents is discussed.

A time hierarchy-based model for kinetics of drug disposition and its use in quantitative structure-activity relationships.

By using the time hierarchy of the processes determining the fate of drugs in biosystems (absorption, transport, distribution, protein binding, and elimination), a one-compartment open model is formulated at a subcellular level for the disposition phase of pharmacokinetics. The resulting disposition function describes the kinetics of the intracellular disposition of drugs as determined by their hydrophobicity, acidity or basicity, affinity to proteins, and rate parameters of elimination. Structure-activity relationships, based on the function with incorporated extrathermodynamic relations, fit the literature data well (fixed-time bioactivity-hydrophobicity profiles, kinetics of microbial degradation of organic compounds, and kinetics of analgesic effects of fentanyl derivatives in rats). Application of the approach, creating a basis for the construction of model-based quantitative structure-time-activity relationship, to biosystems of varying complexity is discussed.

alpha-Amylases and approaches leading to their enhanced stability.

The recent state of the knowledge of properties and structure of alpha-amylases is reviewed with the aim of elucidation the basis for their stabilization. Three principal ways for obtaining stable alpha-amylases (isolation of enzymes from extremophiles, production of extremophilic enzymes in mesophiles, and modification of mesophilic enzymes) are discussed separately. Detailed experimental examples are given for modification approaches.

A kinetic description of the fate of chemicals in biosystems.

A simple kinetic description of the fate of low-molecular-weight compounds in biosystems was derived using the mass action law. Michaelis-Menten kinetics of enzymatic reactions was considered with respect to its two boundary cases, namely first- and zero-order kinetics. Absorption, membrane accumulation, non-covalent protein binding, biotransformation, and excretion have been included in the model, with only the last two steps being considered as time-dependent on the pertinent time scale of hours and days. This time hierarchy allowed for simplification of the resulting expression. In accordance with the results of uptake experiments and contrary to previous approaches, transport of organic molecules into the cell was not considered as the rate-limiting step. The decisive compound properties were found to be hydrophobicity and the intrinsic rate parameters of biotransformation and excretion. The model was applied to the elucidation of the dependence of the observed biotransformation rate parameters on hydrophobicity. The resulting equations are consistent with literature data.