Dancing with oils - the interaction of lipases with different oil/water interfaces.

The use of enzymes as biocatalysts in industrial applications has received much attention during the last few years. Lipases are widely employed in the food and cosmetic industry, for the synthesis of novel biomaterials and as a greener solution for the treatment of waste cooking oils (WCO). The latter topic has been widely explored with the use of enzymes from several origins and types, for the treatment of different used and non-used cooking oils. The experimental conditions of such works are also quite broad, hampering the detailed understanding of the process. In this work we present a detailed characterization of the interaction of several commonly used lipases with different types of vegetal oils and food fats through coarse-grained molecular dynamics simulations. First, the molecular details of the oil/water (O/W) mixtures, namely at the O/W interface, are described. The O/W interface was found to be enriched in triglyceride molecules with higher polarity. Then, the interaction of lipases with oil mixtures is characterized from different perspectives, including the identification of the most important protein residues for this process. The lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML) and Candida antarctica (CALB) were found to bind to the O/W interface in a manner that makes the protein binding site more available for the oil molecules. These enzymes were also found to efficiently bind to the O/W interface of all oil mixtures, which in addition to reactivity factors, may explain the efficient applicability of these enzymes to a large variety of edible oils and WCO.

Effect of Protein Flexibility from Coarse-Grained Elastic Network Parameterizations on the Calculation of Free Energy Profiles of Ligand Binding.

The characterization of the affinity and binding mechanism of specific molecules to a protein active site is scientifically and industrially relevant for many applications. In principle, this information can be obtained using molecular dynamics (MD) simulations by calculating the free energy profile of the process. However, this is a computationally demanding calculation. Currently, coarse-grained (CG) force fields are very well implemented for MD simulations of biomolecular systems. These computationally efficient force fields are a major advantage to the study of large model systems and/or those requiring long simulation times. The Martini model is currently one of the most popular CG force fields for these systems. For the specific case of protein simulations, to correctly maintain the macromolecular three-dimensional structure, the Martini model needs to include an elastic network (EN). In this work, the effect of protein flexibility, as induced by three EN models compatible with the Martini force field, was tested on the calculation of free energy profiles for protein-ligand binding. The EN models used were ElNeDyn, GoMartini, and GEN. The binding of triolein (TOG) and triacetin (TAG) to a lipase protein (thermomyces lanuginosa lipase-TLL) was used as a case study. The results show that inclusion of greater flexibility in the CG parameterization of proteins is of high importance in the calculation of the free energy profiles of protein-ligand systems. However, care must be taken in order to avoid unjustified large protein deformations. In addition, due to molecular flexibility there may be no absolute need for the center of the ligand to reach the center of the protein-binding site. The calculation of the energy profile to a distance of about 0.5 nm from the active site center can be sufficient to differentiate the affinity of different ligands to a protein.

Spiro-Lactams as Novel Antimicrobial Agents.

INTRODUCTION: Structural modulation of previously identified lead spiro-ß-lactams with antimicrobial activity was carried out. OBJECTIVE: The main objective of this work was to synthesize and evaluate the biological activity of novel spiro-lactams based on previously identified lead compounds with antimicrobial activity. METHODS: The target chiral spiro-γ-lactams were synthesized through 1,3-dipolar cycloaddition reaction of a diazo-γ-lactam with electron-deficient dipolarophiles. In vitro activity against HIV and Plasmodium of a wide range of spiro-ß-lactams and spiro-γ-lactams was evaluated. Among these compounds, one derivative with good anti-HIV activity and two with promising antiplasmodial activity (IC50 < 3.5 µM) were identified. RESULTS: A novel synthetic route to chiral spiro-γ-lactams has been established. The studied ß- and γ- lactams were not cytotoxic, and three compounds with promising antimicrobial activity were identified, whose structural modulation may lead to new and more potent drugs. CONCLUSION: The designed structural modulation of biologically active spiro-ß-lactams involved the replacement of the four-membered ß-lactam ring by a five-membered γ-lactam ring. Although conformational and superimposition computational studies revealed no significant differences between ß- and γ- lactam pharmacophoric features, the studied structural modulation did not lead to compounds with a similar biological profile. The observed results suggest that the ß-lactamic core is a requirement for the activity against both HIV and Plasmodium.