Development of an Efficient G‐Quadruplex‐Stabilised Thrombin‐Binding Aptamer Containing a Three‐Carbon Spacer Molecule

The thrombin‐binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G‐quadruplex‐forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three‐carbon spacer (spacer‐C₃), unlocked nucleic acid (UNA) and 3′‐amino‐modified UNA (amino‐UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G‐quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer‐C₃ introduction at the T7 loop position (TBA‐SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA‐SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.     

Activity‐Based Protein Profiling of Rhomboid Proteases in Liposomes

Although activity‐based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity‐based imaging of rhomboid proteases in membrane environments.     

Development of Rhodesain Inhibitors with a 3‐Bromoisoxazoline Warhead

Novel rhodesain inhibitors were obtained by combining an enantiomerically pure 3‐bromoisoxazoline warhead with a specific peptidomimetic recognition moiety. All derivatives behaved as inhibitors of rhodesain, with low micromolar K_i values. Their activity against the enzyme was found to be paralleled by an in vitro antitrypanosomal activity, with IC₅₀ values in the mid‐micromolar range. Notably, a preference for parasitic over human proteases, specifically cathepsins B and L, was observed.     

5‐Hydroxymethyl‐2′‐Deoxyuridine Residues in the Thrombin Binding Aptamer: Investigating Anticoagulant Activity by Making a Tiny Chemical Modification

We report an investigation into analogues of the thrombin binding aptamer (TBA). Individual thymidines were replaced by the unusual residue 5‐hydroxymethyl‐2′‐deoxyuridine (hmU). This differs from the canonical thymidine by a hydroxyl group on the 5‐methyl group. NMR and CD data clearly indicate that all TBA derivatives retain the ability to fold into the “chair‐like” quadruplex structure. The presence of the hmU residue does not significantly affect the thermal stability of the modified aptamers compared to the parent, except for analogue H9 , which showed a marked increase in melting temperature. Although all TBA analogues showed decreased affinities to thrombin, H3 , H7 , and H9 proved to have improved anticoagulant activities. Our data open up the possibility to enhance TBA biological properties, simply by introducing small chemical modifications.     

Thrombin–Fibrin(ogen) Interactions,Host Defense and Risk of Thrombosis

Fibrinogen is a well-known risk factor for arterial and venous thrombosis. Its function is not restricted to clot formation, however, as it partakes in a complex interplay between thrombin, soluble plasma fibrinogen, and deposited fibrin matrices. Fibrinogen, like thrombin, participates predominantly in hemostasis to maintain vascular integrity, but executes some important pleiotropic effects: firstly, as observed in thrombin generation experiments, fibrin removes thrombin from free solution by adsorption. The adsorbed thrombin is protected from antithrombins, notably α2-macroglobulin, and remains physiologically active as it can activate factors V, VIII, and platelets. Secondly, immobilized fibrinogen or fibrin matrices activate monocytes/macrophages and neutrophils via Mac-1 interactions. Immobilized fibrin(ogen) thereby elicits a pro-inflammatory response with a reciprocal stimulating effect of the immune system on coagulation. In contrast, soluble fibrinogen prohibits recruitment of these immune cells. Thus, while fibrin matrices elicit a procoagulant response, both directly by protecting thrombin and indirectly through the immune system, high soluble fibrinogen levels might protect patients due to its immune diminutive function. The in vivo influence of the ‘protective’ plasma fibrinogen versus the ‘pro-thrombotic’ fibrin matrices on thrombosis should be explored in future research.     

Linear and Cyclic Depsipeptidomimetics with β‐Lactam Cores: A Class of New αvβ3 Integrin Receptor Inhibitors

The α_vβ₃ integrin receptor plays an important role in tumor metastasis and tumor‐induced angiogenesis. The inhibition of this receptor with diverse ligands, antibodies, or cyclic peptides is a promising research field for the treatment of a variety of tumors. The replacement of Phe‐(Me)Val dipeptide by a β‐lactam ring in Cilengitide has led to new products that show higher inhibitory activity than the parent cyclopeptide. In particular, substitution of a peptide bond β‐lactam‐NH‐Asp linkage by a β‐lactam‐O‐Asp ester linkage increases the activity of the new cyclodepsipeptide. In the same way it has been found that open‐chain compounds of the form Asp‐β‐lactam‐Arg can interact with the receptor and inhibit its activity moderately. The integrin inhibitory activity of the synthesized compounds has been established by using the CGH array, a method that appears to be a more reliable trial than the classical adhesion test.     

Ac‐LPFFD‐Th: A Trehalose‐Conjugated Peptidomimetic as a Strong Suppressor of Amyloid‐β Oligomer Formation and Cytotoxicity

The inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Peptide‐based inhibitors, which have been widely investigated, are generally derived from original amyloid sequences. Most interestingly, trehalose, a nonreducing disaccharide of α‐glucose, is effective in preventing the aggregation of numerous proteins. We have determined that the development of hybrid compounds could provide new molecules with improved properties that might synergically increase the potency of their single moieties. In this work, the ability of Ac‐LPFFD‐Th, a C‐terminally trehalose‐conjugated derivative, to slow down the Aβ aggregation process was investigated by means of different biophysical techniques, including thioflavin T fluorescence, dynamic light scattering, ESI‐MS, and NMR spectroscopy. Moreover, we demonstrate that Ac‐LPFFD‐Th modifies the aggregation features of Aβ and protects neurons from Aβ oligomers' toxic insult.     

Diversity‐Oriented Synthesis of Diol‐Based Peptidomimetics as Potential HIV Protease Inhibitors and Antitumor Agents

Peptidomimetic HIV protease inhibitors are an important class of drugs used in the treatment of AIDS. The synthesis of a new type of diol‐based peptidomimetics is described. Our route is flexible, uses d ‐glucal as an inexpensive starting material, and makes minimal use of protection/deprotection cycles. Binding affinities from molecular docking simulations suggest that these compounds are potential inhibitors of HIV protease. Moreover, the antiproliferative activities of compounds 33 a , 35 a , and 35 b on HT‐29, M21, and MCF7 cancer cell lines are in the low micromolar range. The results provide a platform that could facilitate the development of medically relevant asymmetrical diol‐based peptidomimetics.     

Beyond Heparinization: Design of Highly Potent Thrombin Inhibitors Suitable for Surface Coupling

During extracorporeal circulation, when blood comes in contact with artificial surfaces, patients receive a standard treatment with anticoagulants to avoid blood coagulation. Dialysis patients in particular are systemically treated with heparin up to four times a week, causing a high burden for the body. For potential anticoagulant modification of external materials, such as dialysis equipment, a series of highly potent thrombin inhibitors was developed. All inhibitors share the general formula arylsulfonyl‐P3‐Pro‐4‐amidinobenzylamide, where P3 is glycyl or a trifunctional amino acid residue in L ‐configuration. Among this series, several derivatives inhibit thrombin with K_i values of less than 1 nM . Specificity measurements revealed that this inhibitor type is highly specific for thrombin with negligible activity against related trypsin‐like serine proteases. X‐ray analysis of the most potent analogue in complex with thrombin demonstrated that the N‐terminal arylsulfonyl group occupies the aryl binding site, whereas the P3 side chain is directed into the solvent and therefore is well suited for further coupling. Based on their in vitro profile, these inhibitors are suitable candidates for the development of hemocompatible materials with anticoagulant properties.     

Design,Synthesis and Characterization of fMLF‐Mimicking AApeptides

The tripeptide N‐formyl‐Met‐Leu‐Phe (fMLF) is a potent neutrophil chemoattractant and the reference agonist for the G protein‐coupled N‐formylpeptide receptor (FPR). As it plays a very important role in host defense and inflammation, there has been considerable interest in the development of fMLF analogues in the hope of identifying potential therapeutic agents. Herein we report the design, synthesis, and evaluation of AApeptides that mimic the structure and function of fMLF. The lead AApeptides induced calcium mobilization and mitogen‐activated protein kinase (MAPK) signal transduction pathways in FPR‐transfected rat basophilic leukemic (RBL) cells. More intriguingly, at high concentrations, certain AApeptides were more effective than fMLF in the induction of calcium mobilization. Their agonistic activity is further supported by their ability to stimulate chemotaxis and the production of superoxide in HL‐60 cells. Similarly to fMLF, these AApeptides are much more selective towards FPR1 than FPR2. These results suggest that the fMLF‐mimicking AApeptides might emerge as a new class of therapeutic agents that target FPRs.     

Cell‐Permeable β‐Peptide Inhibitors of p53/hDM2 Complexation

Look at what the cat(ionic motif) dragged in! We report a general strategy to increase the cell permeability of β³‐peptides. Introduction of a minimal cationic motif within the folded structure of a high‐affinity β³‐peptide ligand for hDM2 led to molecules with high 3₁₄‐helical structure, high hDM2 affinity and sufficient cell permeability to upregulate p53‐dependent genes in live mammalian cells. Minimally cationic β³‐peptides represent the critical first step towards a class of protease‐resistant peptidomimetics that might modulate intracellular biological pathways.

     

Preparation,Characterization, and Gastrointestinal Digestion of Triterpenol-Loaded Liposomes

Triterpenol is an important phytochemical that is beneficial to humans. However, there are limitations of application due to its poor solubility. Liposomes are increasingly utilized as a delivery system for phytochemicals with poor solubility which is suitable for triterpenol. In the present study, the stability of triterpenol-loaded liposomes (Tri-LP) is demonstrated. Tri-LP stability is observed when the mass ratio of phospholipids to triterpenol is 4:1. Sodium alginate (ALG) and chitosan showed different effects on Tri-LP. Tri-LP coated by ALG (Tri-LP-ALG) is stabilized by H-bonding, while electrostatic interactions can stabilize Tri-LP coated by chitosan (Tri-LP-CH). Tri-LP-ALG is more stable than Tri-LP-CH during storage. The bioaccessibility of Tri-LP is 11%. ALG and chitosan show no influence on bioaccessibility of Tri-LP, but reduce the release rate of triterpenol during digestion. Overall, the findings suggest that Tri-LP showed good promise for future application in functional beverages. Practical Applications: Triterpenol has poor solubility both in oil and water, which greatly limits the functional utilization of triterpene in organisms. Liposomes offer a novel approach to triternenol oral delivery system. It meets the current demands for food health. Liposomes can improve the absorption and utilization of triterpenol in vivo digestion. Many studies and applications of liposomes has carried out in medical field while nutritional supplements with liposomes technology are not common in the market. Triterpenol-loaded liposomes can not only increase applications in phytochemicals with poor absorption but also offer an interesting attempt of liposomes in food development.     

Cyclic RGD‐Containing Functionalized Azabicycloalkane Peptides as Potent Integrin Antagonists for Tumor Targeting

Vitronectin receptors α _v β ₃ and α _v β ₅ have emerged as potential therapeutic targets for the treatment of osteoporosis, restenosis, ocular disease, tumor‐induced angiogenesis, metastasis, and sickle‐cell anemia. Among a collection of compounds, a new potent integrin antagonist was synthesized, and its binding toward the α_vβ₃ and α_vβ₅ receptors was evaluated. This molecule is a suitable candidate as a vector for therapeutics and diagnostics.

     

Synthesis and Biological Evaluation of Papain‐Family Cathepsin L‐Like Cysteine Protease Inhibitors Containing a 1,4‐Benzodiazepine Scaffold as Antiprotozoal Agents

Novel papain‐family cathepsin L‐like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure–activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3‐bromoisoxazoline moiety. Several rhodesain and falcipain‐2 inhibitors displaying single‐digit micromolar or sub‐micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds.