Bis-pyridylethenyl benzene as novel backbone for amyloid-ß binding compounds.

Detection of cerebral ß-amyloid (Aß) by targeted contrast agents is of great interest for in vivo diagnosis of Alzheimer's disease (AD). Partly because of their planar structure several bis-styrylbenzenes have been previously reported as potential Aß imaging agents. However, these compounds are relatively hydrophobic, which likely limits their in vivo potential. Based on their structures, we hypothesized that less hydrophobic bis-pyridylethenylbenzenes may also label amyloid. We synthesized several bis-pyridylethenylbenzenes and tested whether these compounds indeed display improved solubility and lower LogP values, and studied their fluorescent properties and Aß binding characteristics. Bis-pyridylethenylbenzenes showed a clear affinity for Aß plaques on both human and murine AD brain sections. Competitive binding experiments suggested a different binding site than Chrysamine G, a well-known stain for amyloid. With a LogP value between 3 and 5, most bis-pyridylethenylbenzenes were able to enter the brain and label murine amyloid in vivo with the bis(4-pyridylethenyl)benzenes showing the most favorable characteristics. In conclusion, the presented results suggest that bis-pyridylethenylbenzene may serve as a novel backbone for amyloid imaging agents.

Polyfluorinated bis-styrylbenzenes as amyloid-ß plaque binding ligands.

Detection of cerebral ß-amyloid (Aß) by targeted contrast agents remains of great interest to aid the in vivo diagnosis of Alzheimer's disease (AD). Bis-styrylbenzenes have been previously reported as potential Aß imaging agents. To further explore their potency as (19)F MRI contrast agents we synthetized several novel fluorinated bis-styrylbenzenes and studied their fluorescent properties and amyloid-ß binding characteristics. The compounds showed a high affinity for Aß plaques on murine and human brain sections. Interestingly, competitive binding experiments demonstrated that they bound to a different binding site than chrysamine G. Despite their high logP values, many bis-styrylbenzenes were able to enter the brain and label murine amyloid in vivo. Unfortunately initial post-mortem (19)F NMR studies showed that these compounds as yet do not warrant further MRI studies due to the reduction of the (19)F signal in the environment of the brain.

'Inverted' analogs of the antibiotic gramicidin S with an improved biological profile.

A series of gramicidin S derivatives 4-15 are presented that have four ornithine residues as polar protonated side chains and two central hydrophobic amino acids with unaltered turn regions. These peptides were screened against human erthrocytes and our standard panel of Gram negative- and Gram positive bacteria, including four MRSA strains. Based on the antibacterial- and hemolytic data, peptides 13 and 14 have an improved biological profile compared to the clinically applied topical antibiotic gramicidin S.

Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives.

In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the ß-strand regions. This structure is in agreement with a previously reported modeling study of the same molecule. The polar side chain of the additional d-amino acid residue is positioned at the same face of the molecule as the hydrophobic side chains, and we believe that because of this compound 2 is considerably less hydrophobic than extended GS derivatives in which the strand regions are exclusively composed of l-amino acids. Using this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues featuring sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity correlates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to efficiently lyse human red blood cells.

Tuning hydrophobicity of highly cationic tetradecameric Gramicidin S analogues using adamantane amino acids.

Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties.

An adamantyl amino acid containing gramicidin S analogue with broad spectrum antibacterial activity and reduced hemolytic activity.

The cyclic cationic antimicrobial peptide gramicidin S (GS) is an effective topical antibacterial agent that is toxic for human red blood cells (hemolysis). Herein, we present a series of amphiphilic derivatives of GS with either two or four positive charges and characteristics ranging between very polar and very hydrophobic. Screening of this series of peptide derivatives identified a compound that combines effective antibacterial activity with virtually no toxicity within the same concentration range. This peptide acts against both Gram-negative and Gram-positive bacteria, including several MRSA strains, and represents an interesting lead for the development of a broadly applicable antibiotic.

Oligoproline helices as structurally defined scaffolds for oligomeric G protein-coupled receptor ligands.

Oligoprolines (OPs) are used as rigid backbone scaffolds for the design of oligomeric ligands that target specific G protein-coupled receptors. The OPs were designed to vary in length, the position and number of the ligand-functionalized residues incorporated. For all synthesized compounds a typical PP type II helix was evidenced by circular dichroism indicating that decoration of the helix with large ligands did not affect the helical conformation. Pharmacological evaluation revealed that oligomerization of an agonist with the use of an oligoproline scaffold showed an increase in potency when compared to the monomeric counterparts.

Gramicidin S derivatives containing cis- and trans-morpholine amino acids (MAAs) as turn mimetics.

The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D-Phe-Pro two-residue turn motifs in the rigid cyclic beta-hairpin structure of GS was replaced with morpholine amino acids (MAA 2-5), differing in stereochemistry and length of the side-chain. The conformational properties of the thus obtained GS analogues (6-9) was assessed by using NMR spectroscopy and X-ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8, containing the dipeptide isostere trans-MAA 4, has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and -negative bacterial strains is better than the derivatives 6, 7 and 9.

Design, synthesis and evaluation of high-affinity binders for the celiac disease associated HLA-DQ2 molecule.

Celiac disease is caused by uncontrolled CD4 T-cell responses directed to wheat-derived gluten peptides bound to the disease predisposing HLA-DQ molecules. The only available treatment is a life-long gluten-free diet which is complicated by the widespread use of wheat-derived gluten in the food industry. As the binding of gluten-derived peptides is a prerequisite for the induction of the inflammatory T-cell response, blockers that would prevent gluten peptide binding to the HLA-DQ molecules might be used as an alternative to the gluten-free diet. In the present study we have analyzed the binding properties of a set of previously identified natural ligands for HLA-DQ2, the primary disease predisposing allele. An in silico method, Epibase, ranked these peptides and the top one, a peptide with a nine amino acid core FVAEYEPVL, was measured among these peptides as the peptide with the highest binding affinity for HLA-DQ2. In a stepwise approach we subsequently tested the impact of N-terminal extensions and systematic single amino acid substitutions within the core of this peptide which revealed that an N-terminal extension with the tripeptide sequence ADA increased binding affinity 5- to 6-fold. In addition the substitution analysis indicated which amino acids were most preferred at anchor residues in the lead peptide, generally leading to an increase of binding affinity with a factor of 2. Next we tested which combinations of such preferred amino acids yielded the best results. The combined results indicate that a peptide with sequence ADAYDYESEELFAA (core in bold) had superior binding properties. This peptide was chosen as a lead peptide for further optimization with non-natural amino acids at the p1 position, since molecular modeling indicated that none of the natural amino acids is able to optimally occupy the p1 pocket. A set of 8 non-proteinogenic amino acids was designed, synthesized and incorporated in the lead peptide (and in two control peptides) and tested for binding to HLA-DQ2. The results indicate that the effect of the incorporation of these non-proteinogenic amino acids depended on the peptide in which they were incorporated and that the maximum increase in binding affinity obtained was approximately 2-fold. Altogether lead sequences were obtained that have a binding affinity for HLA-DQ2 that is 100- to 200-fold higher compared to that of the gluten-derived peptide that has the highest affinity for HLA-DQ2. Such peptides are candidate lead peptides for further optimization. Our results, however, also indicate that in order to obtain further significant increases in binding affinity alternative approaches will have to be explored.

Design of azidoproline containing gluten peptides to suppress CD4+ T-cell responses associated with celiac disease.

Celiac disease is an intestinal disease caused by intolerance for gluten, a common protein in food. A life-long gluten-free diet is the only available treatment. As it is well established that the interaction between proline-rich gluten derived peptides and the human HLA-DQ2 molecules induces immune responses that lead to disease development, we have now designed a series of gluten peptides in which proline residues were replaced by azidoprolines. These peptides were found to bind to HLA-DQ2 with an affinity similar to that of the natural gluten peptide. Moreover, some of these peptides were found to be non-immunogenic and block gluten induced immune responses. These can thus serve as lead compounds for the development of HLA-DQ2 blocker peptides.