Structural and Biochemical Characterization of the Human Angiogenin-Proliferating Cell Nuclear Antigen Interaction.

The molecular details of the interaction between human angiogenin (hAng) and proliferating cell nuclear antigen (PCNA) have been investigated by isothermal titration calorimetry (ITC), mutagenesis, and NMR spectroscopy. The two proteins were shown to interact directly through immunoprecipitation studies of hAng with PCNA in vitro, and their interaction was quantified by ITC, obtaining information on stoichiometry, enthalpy, entropy, and binding kinetics of the association. The hAng-PCNA association is strong, with a Kd value of 126 nM. The interaction surface was mapped by NMR spectroscopy, indicating participating residues. A structural model for the PCNA-hAng complex was constructed by docking and molecular dynamics simulations based on NMR data. The model was validated by mutating the hAng residues Arg5 and Arg101, which seem critical for the complex formation, to glutamate. ITC experiments showed that the angiogenin variants R5E and R5ER101E displayed 6.5 and 7.8 times higher Kd values, respectively, than that of the native protein, indicating the correctness of the model. The hAng S28AT36AS37A and hAng S28AT36AS37AS87A variants were also tested as positive controls, further supporting the validity of the model. The crystal structures of the hAng variants S28AT36AS37A and S28AT36AS37AS87A showed that the mutations did not cause any significant conformational change. This study presents evidence for the structural mode of the hAng-PCNA interaction, revealing valuable information about the angiogenin and PCNA biological roles in the cytoplasm.

Study of Stability, Cytotoxic and Antimicrobial Activity of Chios Mastic Gum Fractions (Neutral, Acidic) after Encapsulation in Liposomes.

Mastic gum is a resinous sap produced by Pistacia lentiscus growing in the island of Chios (Greece) and has been recognized since Antiquity for its distinctive aroma as well as medical properties (antimicrobial, antioxidant, anti-inflammatory ones). The oral absorption of Chios Mastic gum (an insoluble polymer of poly-ß-myrcene is among the most abundant contents) is poor due to its low water-solubility. We report in this study, two different Chios mastic gum extracts, the acidic mastic gum extract-AMGE-and the neutral one-NMGE, both prepared after removal of the contained polymer in order to ameliorate solubility and enhance in vivo activity. Liposomes are presented as a promising delivery system due to their physicochemical and biophysical properties to increase stability and absorption efficiency of the mastic gum extracts within the gastrointestinal (GI) tract. The aim of this study was to evaluate the stability in GI simulated conditions together with cytotoxic and antimicrobial activity of the two extracts (AMGE and NMGE) after encapsulation in a well characterized liposome formulation. Liposomes-AMGE complex showed an improved stability behavior in GI simulated conditions. Both assayed extracts showed significant dose dependent inhibition against the growth of liver cancer HepG2 cells and an interesting antimicrobial activity against several microorganisms. Conclusively, encapsulation could be evaluated as a beneficial procedure for further applications of mastic resin.

Design and Synthesis of Type-IV Inhibitors of BRAF Kinase That Block Dimerization and Overcome Paradoxical MEK/ERK Activation.

Despite the clinical success of BRAF inhibitors like vemurafenib in treating metastatic melanoma, resistance has emerged through "paradoxical MEK/ERK signaling" where transactivation of one protomer occurs as a result of drug inhibition of the other partner in the activated dimer. The importance of the dimerization interface in the signaling potential of wild-type BRAF in cells expressing oncogenic Ras has recently been demonstrated and proposed as a site of therapeutic intervention in targeting cancers resistant to adenosine triphosphate competitive drugs. The proof of concept for a structure-guided approach targeting the dimerization interface is described through the design and synthesis of macrocyclic peptides that bind with high affinity to BRAF and that block paradoxical signaling in malignant melanoma cells occurring through this drug target. The lead compounds identified are type-IV kinase inhibitors and represent an ideal framework for conversion into next-generation BRAF inhibitors through macrocyclic drug discovery.

In vitro and in vivo assessment of vitamin A encapsulation in a liposome-protein delivery system.

Vitamin A (VA) is an essential nutrient needed in small amounts by humans and supports a wide range of biological actions. Retinol, the most common and most biologically active form of VA has also been found to inhibit peroxidation processes in membranes and it has been widely used as an ingredient with pharmaceutical and nutritional applications. VA is a lipophilic molecule, sensitive to air, oxidizing agents, ultraviolet light and low pH levels. For these reasons, it is necessary for VA to be protected against oxidation. Another disadvantage in the application of VA is its low solubility in aqueous media. Both issues (sensitivity and solubility) can be solved by employing encapsulation techniques. Liposomes can efficiently encapsulate lipid-soluble materials, such as VA. The encapsulated materials are protected from environmental and chemical changes. A new liposome/ß-lactoglobulin formulation has been developed as a stable delivery system for VA. The aim of this study was the encapsulation of VA into ß-lactoglobulin-liposome complexes, recently developed in our laboratory. The in vivo bioavailability characterization of VA was tested after administration in laboratory animals (mice). In this report, we demonstrate that VA could be efficiently entrapped and delivered in a phospholipid-sterol-protein membrane resembling system, a newly synthesized promising carrier. Based on this finding, the phospholipid-sterol-protein membrane resembling system may be one of the promising approaches to enhance VA absorption and to overcome the formulation difficulties associated with lipophilic means. The carrier system described here has huge potential in food fortification applications to treat VA deficiency.

Thermodynamic, crystallographic and computational studies of non-mammalian fatty acid binding to bovine ß-Lactoglobulin.

The milk protein ß-lactoglobulin has been widely studied since its discovery, both as a purified protein and in mixtures with other milk proteins, where its effect on the processing properties is of importance to the dairy industry. The protein can bind a variety of small hydrophobic molecules, which may allow its use as an oral delivery vehicle. In the present study we have examined the binding of odd-numbered fatty acids by isothermal calorimetry (ITC), X-ray crystallography and computer modelling to provide a clearer picture of the extent and variability of the central binding pocket. The Kd values for the fatty acids C13, C15, C16, C17 and C19 as determined by ITC are 1.93, 2.91, 3.05, 4.11 and 8.67 × 10-7 M, respectively. The molecular structures revealed the ligands bound in the central cavity with generally well ordered lipophilic tails but significant positional variation at the carboxyl group end. In silico docking analyses identified the lipophilic interactions within the central cavity as the main driving force for binding with electrostatic interactions and H-bonds playing a minor role.

ß-Lactoglobulin improves liposome's encapsulation properties for vitamin E delivery.

Vitamin E (VE) or α-tocopherol is the major fat-soluble antioxidant in the human body. It is a sensitive, easily oxidized in the air, molecule, so it must be protected from pro-oxidant elements which could affect its physiological benefits. Encapsulation constitutes a promising approach to maintain VE native properties over time and increase its concentration in aqueous media. Liposomes have been studied as sustained delivery systems, being biodegradable, non-toxic and non-immunogenic. A new liposome/ß-lactoglobulin (ß-Lg) formulation has been developed and characterized as a possible stable delivery system for VE. ß-Lg has been selected due to its property to bind a variety of hydrophobic molecules. The aim of this study was the preparation of ß-Lg-liposome formulation and the determination of VE encapsulation efficiency, in order to develop a new more efficient carrier for VE in aqueous media.