Design, Synthesis and Catalytic Activity of Protein Containing Thiotyrosine as an Active Site Residue.

Native chemical ligation is a key reaction in the toolbox of chemical methods for the synthesis of native and modified proteins. The catalysis of ligation is commonly performed by using small aryl-thiol molecules added at high concentrations. In this work, we incorporated thiotyrosine, a non-canonical amino acid containing an aryl-thiol moiety, into a designed cyclic protein « sans queue ni tête ¼. Importantly, the protein environment reduced the pKa of the thiol group to 5.8-5.9, which is significantly lower than the previously reported value for thiotyrosine in a short peptide (pKa 6.4). Furthermore, we demonstrated the catalytic activity of this protein both as hydrolase and in native chemical ligation of peptides. These results will be useful for the development of efficient protein catalysts (enzymes) for protein synthesis and modification.

Redesigning of Cell-Penetrating Peptides to Improve Their Efficacy as a Drug Delivery System.

Cell-penetrating peptides (CPP) are promising tools for the transport of a broad range of compounds into cells. Since the discovery of the first members of this peptide family, many other peptides have been identified; nowadays, dozens of these peptides are known. These peptides sometimes have very different chemical-physical properties, but they have similar drawbacks; e.g., non-specific internalization, fast elimination from the body, intracellular/vesicular entrapment. Although our knowledge regarding the mechanism and structure-activity relationship of internalization is growing, the prediction and design of the cell-penetrating properties are challenging. In this review, we focus on the different modifications of well-known CPPs to avoid their drawbacks, as well as how these modifications may increase their internalization and/or change the mechanism of penetration.

Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers.

Cell-penetrating peptides represent an emerging class of carriers capable of effective cellular delivery. This work demonstrates the preparation and investigation of efficient CPPs. We have already shown that the presence of 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and Trp greatly increase the uptake of oligoarginines. This work is a further step in that direction. We have explored the possibility of employing unnatural, aromatic amino acids, to mimic Trp properties and effects. The added residues allow the introduction of aromaticity, not as a side-chain group, but rather as a part of the sequence. The constructs presented exceptional internalization on various cell lines, with an evident structure-activity relationship. The CPPs were investigated for their entry mechanisms, and our peptides exploit favorable pathways, yet one of the peptides relies highly on direct penetration. Confocal microscopy studies have shown selectivity towards the cell lines, by showing diffuse uptake in FADU cells, while vesicular uptake takes place in SCC-25 cell line. These highly active CPPs have proved their applicability in cargo delivery by successfully delivering antitumor drugs into MCF-7 and MDA-MB-231 cells. The modifications in the sequences allow the preparation of short yet highly effective constructs able to rival the penetration of well-known CPPs such as octaarginine (Arg8).

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4-((4-(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.

Modulators of calpain activity: inhibitors and activators as potential drugs.

Introduction: Calpains are intracellular Ca2+-dependent cysteine proteases with 15 known members in the enzyme family. They act as regulatory enzymes, their cleavage modifying the function of their substrates. As their substrates have important roles in many physiological processes, adequate function of calpains is mandatory for normal cellular functions. The adverse operation of them is often related to diseases (e.g. neurodegenerative disorders, cancer, type 2 diabetes mellitus, or limb-girdle muscular dystrophy type 2A).Areas covered: Herein, the authors give an overview of calpains, their structure as well as their physiological and pathological functions. The authors further consider the challenges in calpain inhibitor and activator drug discovery and summarize examples of good candidates. New and applicable strategies are also discussed.Expert opinion: Calpain enzymes are attractive targets for inhibitor or activator design and development. The authors believe this area of research is of high potential, although it has many challenges. For one, the selective and targeted inhibition or activation of calpains is needed. Furthermore, uncontrolled calpain activation may cause more serious side effects and so caution is needed when designing novel therapeutics.