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BMP‐1/tolloid‐like proteinases belong to the astacin family of human metalloproteinases, together with meprins and ovastacin. They represent promising targets to treat or prevent a wide range of diseases such as fibrotic disorders or cancer. However, the study of their pathophysiological roles is still impaired by the lack of well‐characterized inhibitors and the questions that remain regarding their selectivity and in vivo efficiency. As a first step towards the identification of suitable tools to be used in functional studies, we have undertaken a systematic comparison of seven molecules known to affect the proteolytic activity of human astacins including three hydroxamates (FG‐2575, UK383,367, S33A), the protein sizzled, a new phosphinic inhibitor (RXP‐1001) and broad‐spectrum protease inhibitors (GM6001, actinonin). Their efficacy in vitro, their cellular toxicity and efficacy in cell cultures were thoroughly characterized. We found that these molecules display very different potency and selectivity profiles, with hydroxamate FG‐2575 and the protein sizzled being very powerful and selective inhibitors of BMP‐1, whereas phosphinic peptide RXP‐1001 behaves as a broad‐spectrum inhibitor of astacins. Their use should therefore be carefully considered in agreement with the aim of the study to avoid result misinterpretation. BMP‐1/tolloid‐like proteinases (BTPs) are prominent members of the astacin subgroup of metalloproteinases which are currently evaluated as therapeutic targets in several diseases. We report a detailed analysis of seven potential BTP inhibitors, both synthetic and natural molecules, in terms of potency, toxicity and selectivity towards meprins. This study emphasises the importance of carefully choosing the best tool for each application. [ABSTRACT FROM AUTHOR]

Small RNA molecules such as microRNA and small interfering RNA (siRNA) have become promising therapeutic agents because of their specificity and their potential to modulate gene expression. Any gene of interest can be potentially up- or down-regulated, making RNA-based technology the healthcare breakthrough of our era. However, the functional and specific delivery of siRNAs into tissues of interest and into the cytosol of target cells remains highly challenging, mainly due to the lack of efficient and selective delivery systems. Among the variety of carriers for siRNA delivery, peptides have become essential candidates because of their high selectivity, stability, and conjugation versatility. Here, we describe the development of molecules encompassing siRNAs against SOD1 , conjugated to peptides that target the low-density lipoprotein receptor (LDLR), and their biological evaluation both in vitro and in vivo.

Neurotensin (NT) acts as a primary neurotransmitter and neuromodulator in the CNS and has been involved in a number of CNS pathologies including epilepsy. NT mediates its central and peripheral effects by interacting with the NTSR1, NTSR2, and Sort1/NTSR3 receptor subtypes. To date, little is known about the precise expression of the NT receptors in brain neural cells and their regulation in pathology. In the present work, we studied the cellular distribution of the NTSR2 protein in the rat hippocampus and questioned whether its expression was modulated in conditions of neuroinflammation using a model of temporal lobe epilepsy induced by pilocarpine. This model is characterized by a rapid and intense inflammatory reaction with reactive gliosis in the hippocampus. We show that NTSR2 protein is expressed in hippocampal astrocytes and its expression increases together with astrocyte reactivity following induction of status epilepticus. NTSR2 immunoreactivity is also increased in astrocytes proximal to blood vessels and their end-feet, and in endothelial cells. Proinflammatory factors such as IL1β and LPS induced NTSR2 mRNA and protein in cultured astroglial cells. Antagonizing NTSR2 with SR142948A decreased NTSR2 expression as well as astroglial reactivity. Together, our results suggest that NTSR2 is implicated in astroglial and gliovascular inflammation and that targeting the NTSR2 receptor may open new avenues in the regulation of neuroinflammation in CNS diseases., (© 2021 Wiley Periodicals LLC.)

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells-or organs that express the LDLR.

The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals ( ldlr -/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier., (© FASEB.)

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC] c DPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC] c ) which specifically binds hLDLR with a K D of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC] c ), which showed the highest K D value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 10 6 s -1. M -1 range, and off-rates varying from the low 10 -2 s -1 to the 10 -1 s -1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"] c ), showing a K D of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3 H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.

The internalization of cell-penetrating peptides (CPPs) into liposomes (large unilamellar vesicles, LUVs) was studied with a rapid and robust procedure based on the quenching of a small fluorescent probe, 7-nitrobenz-2-oxa-1,3-diazole (NBD). Quenching can be achieved by reduction with dithionite or by pH jump. LUVs with different compositions of phospholipids (PLs) were used to screen the efficacy of different CPPs. In order to "validate" the composition of the membrane models, a control cationic peptide, which does not enter eukaryotic cells, was included in the study. It was found that pure DOPG or DOPG within ternary mixtures with cholesterol are the most appropriate models for studying CPP translocation. An anionic lipid, such as DOPG, is required for the adsorption of the basic peptides on the surface of LUVs. In addition, it acts as transfer agent through the lipid bilayer. A fluid phase and/or the presence of phase defects also appear mandatory for the internalization to occur. The neutralization of charges within an inverted micelle demonstrated in the case of DOPG and also proposed for a ternary mixture of PLs might not be the only mechanism for the CPP translocation. Finally, it is shown that oleic acid facilitates the entry inside LUVs in gel phase of a series of cationic peptides including CPPs and also the negative control peptide PKCi., (© 2015 Wiley Periodicals, Inc.)

BMP-1/tolloid-like proteinases (BTPs) are major enzymes involved in extracellular matrix assembly and activation of bioactive molecules, both growth factors and anti-angiogenic molecules. Although the control of BTP activity by several enhancing molecules is well established, the possibility that regulation also occurs through endogenous inhibitors is still debated. Secreted frizzled-related proteins (sFRPs) have been studied as possible candidates, with highly contradictory results, after the demonstration that sizzled, a sFRP found in Xenopus and zebrafish, was a potent inhibitor of Xenopus and zebrafish tolloid-like proteases. In this study, we demonstrate that mammalian sFRP-1, -2, and -4 do not modify human BMP-1 activity on several of its known substrates including procollagen I, procollagen III, pN-collagen V, and prolysyl oxidase. In contrast, Xenopus sizzled appears as a tight binding inhibitor of human BMP-1, with a K(i) of 1.5 ± 0.5 nM, and is shown to strongly inhibit other human tolloid isoforms mTLD and mTLL-1. Because sizzled is the most potent inhibitor of human tolloid-like proteinases known to date, we have studied its mechanism of action in detail and shown that the frizzled domain of sizzled is both necessary and sufficient for inhibitory activity and that it acts directly on the catalytic domain of BMP-1. Residues in sizzled required for inhibition include Asp-92, which is shared by sFRP-1 and -2, and also Phe-94, Ser-43, and Glu-44, which are specific to sizzled, thereby providing a rational basis for the absence of inhibitory activity of human sFRPs.

Glycosylated cell penetrating peptides (CPPs) have been conjugated to a peptide cargo and the efficiency of cargo delivery into wild type Chinese hamster ovary (CHO) and proteoglycan deficient CHO cells has been quantified by MALDI-TOF mass spectrometry and compared to tryptophan- or alanine containing CPPs. In parallel, the behavior of these CPPs in contact with model membranes has been characterized by different biophysical techniques: Differential Scanning and Isothermal Titration Calorimetries, Imaging Ellipsometry and Attenuated Total Reflectance IR spectroscopy. With these CPPs we have demonstrated that tryptophan residues play a key role in the insertion of a CPP and its conjugate into the membrane: galactosyl residues hampered the internalization when introduced in the middle of the amphipathic secondary structure of a CPP but not when added to the N-terminus, as long as the tryptophan residues were still present in the sequence. The insertion of these CPPs into membrane models was enthalpy driven and was related to the number of tryptophans in the sequence of these secondary amphipathic CPPs. Additionally, we have observed a certain propensity of the investigated CPP analogs to aggregate in contact with the lipid surface., (Copyright © 2011 Elsevier B.V. All rights reserved.)