Synthesis of Novel Arsonolipids and Development of Novel Arsonoliposome Types.

Arsonolipids represent a class of arsenic-containing compounds with interesting biological properties either as monomers or as nanostructure forming components, such as arsonoliposomes that possess selective anticancer activity as proven by in vitro and in vivo studies. In this work, we describe, for the first time, the synthesis of novel arsono-containing lipids where the alkyl groups are connected through stable ether bonds. It is expected that this class of arsonolipids, compared with the corresponding ester linked, will have higher chemical stability. To accomplish this task, a new methodology of general application was developed, where a small arsono compound, 2-hydroxyethylarsonic acid, when protected with thiophenol, can be used in an efficient and simple way as a building block for the synthesis of arsono-containing lipids as well as other arsono-containing biomolecules. Thus, besides the above-mentioned arsonolipid, an arsono cholesterol derivative was also obtained. Both ether arsonolipid and arsono cholesterol were able to form liposomes having similar physicochemical properties and integrity to conventional arsonoliposomes. Furthermore, a preliminary in vitro anticancer potential assessment of the novel ether arsonolipid containing liposomes against human prostate cancer (PC-3) and Lewis lung carcinoma (LLC) cells showed significant activity (dose- and time-dependent), which was similar to that of the conventional arsonoliposomes (studied before). Given the fact that novel arsonolipids may be more stable compared to the ones used in conventional arsonoliposomes, the current results justify further exploitation of the novel compounds by in vitro and in vivo studies.

Generation of a quenched phosphonate activity-based probe for labelling the active KLK7 protease.

Kallikrein 7 (KLK7) is a chymotrypsin-like serine protease with established roles in skin diseases like the rare Netherton syndrome, an overdesquamating and inflammatory condition, but also common atopic dermatitis, and a potential drug target for these and possibly other diseases. Nevertheless, tools to determine the active KLK7 enzyme are not available. Here, a mixed alkyl aryl phosphonate quenched activity-based probe that detects the active KLK7 was developed and evaluated in vitro. This KLK7-qABP can potentially be used to monitor KLK7 activity in vivo.

A novel theranostic activity-based probe targeting kallikrein 7 for the diagnosis and treatment of skin diseases.

We applied a new in silico approach for using protease-substrate motifs to design a kallikrein 7 (KLK7)-specific phosphonate activity-based probe (ABP) to quantify the active KLK7 in situ. Epidermal application of the ABP-inhibitor on Spink5-/-Klk5-/- mice, a Netherton syndrome model, reversed disease hallmarks, providing preclinical proof-of-concept for using ABPs as theranostics.

Plausible Emergence of Biochemistry in Enceladus Based on Chemobrionics.

Saturn's satellite Enceladus is proposed to have a soda-type subsurface ocean with temperature able to support life and an iron ore-based core. Here, it was demonstrated that ocean chemistry related to Enceladus can support the development of Fe-based hydrothermal vents, one of the places suggested to be the cradle of life. The Fe-based chemical gardens were characterized with Fourier-transform (FT)IR spectroscopy and XRD. The developed chemobrionic structures catalyzed the condensation polymerization of simple organic prebiotic molecules to kerogens. Further, they could passively catalyze the condensation of the prebiotic molecule formamide to larger polymers, suggesting that elementary biochemical precursors could have emerged in Enceladus.

Discovery of a stable tripeptide targeting the N-domain of CRF1 receptor.

The corticotropin-releasing factor (CRF) and its CRF1 receptor (CRF1R) play a central role in the maintenance of homeostasis. Malfunctioning of the CRF/CRF1R unit is associated with several disorders, such as anxiety and depression. Non-peptide CRF1R-selective antagonists have been shown to exert anxiolytic and antidepressant effects on experimental animals. However, none of them is in clinical use today because of several side effects, thus demonstrating the need for the development of other more suitable CRF1R antagonists. In an effort to develop novel CRF1R antagonists we designed, synthesized and chemically characterized two tripeptide analogues of CRF, namely (R)-LMI and (S)-LMI, having their Leu either in R (or D) or in S (or L) configuration, respectively. Their design was based on the crystal structure of the N-extracellular domain (N-domain) of CRF1R/CRF complex, using a relevant array of computational methods. Experimental evaluation of the stability of synthetic peptides in human plasma has revealed that (R)-LMI is proteolytically more stable than (S)-LMI. Based on this finding, (R)-LMI was selected for pharmacological characterization. We have found that (R)-LMI is a CRF antagonist, inhibiting (1) the CRF-stimulated accumulation of cAMP in HEK 293 cells expressing the CRF1R, (2) the production of interleukins by adipocytes and (3) the proliferation rate of RAW 264.7 cells. (R)-LMI likely blocked agonist actions by interacting with the N-domain of CRF1R as suggested by data using a constitutively active chimera of CRF1R. We propose that (R)-LMI can be used as an optimal lead compound in the rational design of novel CRF antagonists.

Biologically relevant conformational features of linear and cyclic proteolipid protein (PLP) peptide analogues obtained by high-resolution nuclear magnetic resonance and molecular dynamics.

Proteolipid protein (PLP) is one of the main proteins of myelin sheath that are destroyed during the progress of multiple sclerosis (MS). The immunodominant PLP139-151 epitope is known to induce experimental autoimmune encephalomyelitis (EAE, animal model of MS), wherein residues 144 and 147 are recognized by T cell receptor (TCR) during the formation of trimolecular complex with peptide-antigen and major histocompability complex. The conformational behavior of linear and cyclic peptide analogues of PLP, namely PLP139-151 and cyclic (139-151) (L144, R147) PLP139-151, have been studied in solution by means of nuclear magnetic resonance (NMR) methods in combination with unrestrained molecular dynamics simulations. The results indicate that the side chains of mutated amino acids in the cyclic analogue have different spatial orientation compared with the corresponding side chains of the linear analogue, which can lead to reduced affinity to TCR. NMR experiments combined with theoretical calculations pave the way for the design and synthesis of potent restricted peptides of immunodominant PLP139-151 epitope as well as non peptide mimetics that rises as an ultimate goal.

A novel synthetic luteinizing hormone-releasing hormone (LHRH) analogue coupled with modified ß-cyclodextrin: insight into its intramolecular interactions.

BACKGROUND: Cyclodextrins (CDs) in combination with therapeutic proteins and other bioactive compounds have been proposed as candidates that show enhanced chemical and enzymatic stability, better absorption, slower plasma clearance and improved dose-response curves or immunogenicity. As a result, an important number of therapeutic complexes between cyclodextrins and bioactive compounds capable to control several diseases have been developed. RESULTS: In this article, the synthesis and the structural study of a conjugate between a luteinizing hormone-releasing hormone (LHRH) analogue, related to the treatment of hormone dependent cancer and fertility, and modified ß-cyclodextrin residue are presented. The results show that both the phenyl group of tyrosine (Tyr) as well as the indole group of tryptophan (Trp) can be encapsulated inside the cyclodextrin cavity. Solution NMR experiments provide evidence that these interactions take place intramolecularly and not intermolecularly. CONCLUSIONS: The study of a LHRH analogue conjugated with modified ß-cyclodextrin via high field NMR and MD experiments revealed the existence of intramolecular interactions that could lead to an improved drug delivery. GENERAL SIGNIFICANCE: NMR in combination with MD simulation is of great value for a successful rational design of peptide-cyclodextrin conjugates showing stability against enzymatic proteolysis and a better pharmacological profile.