Pharmacological characterization of a novel peptide inhibitor of the Keap1-Nrf2 protein-protein interaction.

LAS200813 is a novel bicyclic lipopeptide that activates Nrf2 by binding to Keap1, thereby antagonising the Keap1-Nrf2 protein-protein interaction. In this work we report the pharmacological characterization of LAS200813 in Nrf2-dependent translational preclinical models. LAS200813 binds to Keap1 with high affinity (IC50: 0.73 nM) and is able to induce the translocation of Nrf2 to the nucleus. Furthermore, LAS200813 increases the expression of Nrf2 target genes in human bronchial epithelial cells (EC50 of 96 and 70 nM for srxn1 and nqo1, respectively). Similarly, the intratracheal administration of LAS200813 to rats increases the expression of Nrf2-dependent genes in lung tissue, an effect that lasts for a few hours. Moreover, in cells exposed to cigarette smoke, LAS200813 shows an antioxidant effect by increasing the production of glutathione and prevents cellular apoptosis. In conclusion, the results described herein demonstrate that LAS200813 is a potent non-electrophilic Nrf2-activating peptide designed to be administered by inhaled route which may be a potential therapeutic strategy for respiratory diseases driven by oxidative stress.

The road to the synthesis of "difficult peptides".

The last decade has witnessed a renaissance of peptides as drugs. This progress, together with advances in the structural behavior of peptides, has attracted the interest of the pharmaceutical industry in these molecules as potential APIs. In the past, major peptide-based drugs were inspired by sequences extracted from natural structures of low molecular weight. In contrast, nowadays, the peptides being studied by academic and industrial groups comprise more sophisticated sequences. For instance, they consist of long amino acid chains and show a high tendency to form aggregates. Some researchers have claimed that preparing medium-sized proteins is now feasible with chemical ligation techniques, in contrast to medium-sized peptide syntheses. The complexity associated with the synthesis of certain peptides is exemplified by the so-called "difficult peptides", a concept introduced in the 80's. This refers to sequences that show inter- or intra-molecular ß-sheet interactions significant enough to form aggregates during peptide synthesis. These structural associations are stabilized and mediated by non-covalent hydrogen bonds that arise on the backbone of the peptide and-depending on the sequence-are favored. The tendency of peptide chains to aggregate is translated into a list of common behavioral features attributed to "difficult peptides" which hinder their synthesis. In this regard, this manuscript summarizes the strategies used to overcome the inherent difficulties associated with the synthesis of known "difficult peptides". Here we evaluate several external factors, as well as methods to incorporate chemical modifications into sequences, in order to describe the strategies that are effective for the synthesis of "difficult peptides". These approaches have been classified and ordered to provide an extensive guide for achieving the synthesis of peptides with the aforementioned features.

Formylation of electron-rich aromatic rings mediated by dichloromethyl methyl ether and TiCl4: scope and limitations.

Here the aromatic formylation mediated by TiCl4 and dichloromethyl methyl ether previously described by our group has been explored for a wide range of aromatic rings, including phenols, methoxy- and methylbenzenes, as an excellent way to produce aromatic aldehydes. Here we determine that the regioselectivity of this process is highly promoted by the coordination between the atoms present in the aromatic moiety and those in the metal core.

Semipermanent C-terminal carboxylic acid protecting group: application to solubilizing peptides and fragment condensation.

The 2-methoxy-4-methylsulfinylbenzyl alcohol (Mmsb-OH) safety-catch linker has been described as a useful tool to overcome two obstacles in peptide synthesis: the solubility and fragment condensation of peptides. The incorporation of the linker into an insoluble peptide target, thereby allowing the conjugation of a poly-Lys as a "solubilizing tag", notably enhanced the solubility of the peptide. The selective conditions that remove that linker favored its incorporation as a semipermanent C-terminal protecting group, thereby allowing fragment condensation of peptides.

2-Methoxy-4-methylsulfinylbenzyl: a backbone amide safety-catch protecting group for the synthesis and purification of difficult peptide sequences.

The use of 2-methoxy-4-methylsulfinylbenzyl (Mmsb) as a new backbone amide-protecting group that acts as a safety-catch structure is proposed. Mmsb, which is stable during the elongation of the sequence and trifluoroacetic acid-mediated cleavage from the resin, improves the synthetic process as well as the properties of the quasi-unprotected peptide. Mmsb offers the possibility of purifying and characterizing complex peptide sequences, and renders the target peptide after NH4 I/TFA treatment and subsequent ether precipitation to remove the cleaved Mmsb moiety. First, the "difficult peptide" sequence H-(Ala)10-NH2 was selected as a model to optimize the new protecting group strategy. Second, the complex, bioactive Ac-(RADA)4-NH2 sequence was chosen to validate this methodology. The improvements in solid-phase peptide synthesis combined with the enhanced solubility of the quasi-unprotected peptides, as compared with standard sequences, made it possible to obtain purified Ac-(RADA)4-NH2. To extend the scope of the approach, the challenging Aß(1-42) peptide was synthesized and purified in a similar manner. The proposed Mmsb strategy opens up the possibility of synthesizing other challenging small proteins.

Linear versus branched poly-lysine/arginine as polarity enhancer tags.

The design and synthesis of Lys- and Arg-containing peptides as solubilizing tags were studied to evaluate their influence on polarity. The relevance of spatial arrangement of polar groups, in α- or ε-amino positions, was confirmed by chromatographic analysis of a rational PolyLys-based synthesized structure. The most promising of the solubilizing tags here analyzed was conjugated to a commercial water-insoluble drug (indomethacin) as proof of concept.

Optimized Fmoc solid-phase synthesis of the cysteine-rich peptide linaclotide.

Linaclotide, a small 14-mer peptide highly rich in cysteines, is currently in phase III clinical trials for the treatment of gastrointestinal disorders. The challenge in the assembly of linaclotide consists of achieving the correct and clean folding of its three disulfide bridges. For this purpose, a number of regioselective, semiregioselective, and random strategies have been studied. In addition to selecting distinct protecting groups for the thiol function, their position in the sequence, the influence of the neighboring protecting groups, as well as the order in which the disulfides fold were studied. Here we describe an optimized solid-phase synthesis of linaclotide that should allow the production of this peptide in multigram amounts.

ChemMatrix(®) for complex peptides and combinatorial chemistry.

CM resin is a totally PEG-based resin, made exclusively from primary ether bonds and therefore highly chemically stable. Compared to other PEG resins, it exhibits good loading and is user friendly because of its free-flowing form upon drying. It shows improved performance over PS resins for the preparation of hydrophobic, highly structured poly-Arg peptides. In combination with ψPros, it allows the synthesis of small proteins such as the chemokine RANTES. Like other PEG-based resins, CM resin swells well in biocompatible solvents such as water, thereby allowing on-bead screening. Furthermore, the high loading of this resin permits the use of a tiny quarter of a bead as a microreactor for HPLC and MALDI-TOF analysis, thus further extending its applications in the field of combinatorial chemistry.

Solid-phase peptide synthesis using acetonitrile as a solvent in combination with PEG-based resins.

This manuscript shows that ACN can be an excellent choice for the coupling of hindered amino acids as illustrated by the coupling of Fmoc-amino acids on free amino acids anchored on a BAL synthesis. Furthermore, ACN can be a good alternative for solid-phase peptide synthesis in the absence of DMF (washings, removal of Fmoc, and coupling).

From the one-bead-one-compound concept to one-bead-one-reactor.

The one-bead-one-compound method gives access to millions of compounds that can be screened directly on the bead. Although characterization techniques are increasingly potent and reliable, problems can still be encountered in deciphering the sequence of the active compound because of sensitiveness or manipulation of the bead. ChemMatrix, a totally PEG-based resin, has resolved the synthesis of peptides of outstanding difficulty. Like other PEG-based resins, it permits on-bead screening because of its compatibility in aqueous media and has the further advantage of having a high loading, comparable to polystyrene resins. ChemMatrix beads previously swelled in water can be nicely divided into four parts that can be characterized using different analytical techniques or just stored for safety or for further testing. The four bead parts show high homogeneity and can thus be considered to be replicas.