Chemical modulation of peptoids: synthesis and conformational studies on partially constrained derivatives.

The high conformational flexibility of peptoids can generate problems in biomolecular selectivity as a result of undesired off-target interactions. This drawback can be counterbalanced by restricting the original flexibility to a certain extent, thus leading to new peptidomimetics. By starting from the structure of an active peptoid as an apoptosis inhibitor, we designed two families of peptidomimetics that bear either 7-substituted perhydro-1,4-diazepine-2,5-dione 2 or 3-substituted 1,4-piperazine-2,5-dione 3 moieties. We report an efficient, solid-phase-based synthesis for both peptidomimetic families 2 and 3 from a common intermediate. An NMR spectroscopic study of 2a,b and 3a,b showed two species in solution in different solvents that interconvert slowly on the NMR timescale. The cis/trans isomerization around the exocyclic tertiary amide bond is responsible for this conformational behavior. The cis isomers are more favored in nonpolar environments, and this preference is higher for the six-membered-ring derivative 3a,b. We propose that the hydrogen-bonding pattern could play an important role in the cis/trans equilibrium process. These hydrogen bonds were characterized in solution, in the solid state (i.e., by using X-ray studies), and by molecular modeling of simplified systems. A comparative study of a model peptoid 10 containing the isolated tertiary amide bond under study outlined the importance of the heterocyclic moiety for the prevalence of the cis configuration in 2a and 3a. The kinetics of the cis/trans interconversion in 2a, 3a, and 10 was also studied by variable-temperature NMR spectroscopic analysis. The full line-shape analysis of the NMR spectra of 10 revealed negligible entropic contribution to the energetic barrier in this conformational process. A theoretical analysis of 10 supported the results observed by NMR spectroscopic analysis. Overall, these results are relevant for the study of the peptidomimetic/biological-target interactions.

Conjugation of a novel Apaf-1 inhibitor to peptide-based cell-membrane transporters: effective methods to improve inhibition of mitochondria-mediated apoptosis.

We have identified a family of peptoids that inhibits in vitro the activity of the apoptosome, a macromolecular complex that activates mitochondrial-dependent apoptosis pathways. The analysis of peptide-based cell compatible delivery systems of the most active peptoid is presented. The active peptoid was then fused to cell penetrating peptides (CPP) as penetratin (PEN-peptoid) and HIV-1 TAT (TAT-peptoid). PEN-peptoid showed greater cell viability and as a consequence better efficiency as an apoptosis inhibitor than the TAT-peptoid. The intracellular trafficking of both inhibitors was studied by flow cytometry and confocal fluorescence microscopy. Finally, the influence of the cargo (peptoid) molecules on the conformational behavior of the CPP in buffers and in membrane mimetic environments was analyzed using circular dichroism (CD) spectroscopy.

Neurotoxicity of prion peptides mimicking the central domain of the cellular prion protein.

The physiological functions of PrP(C) remain enigmatic, but the central domain, comprising highly conserved regions of the protein may play an important role. Indeed, a large number of studies indicate that synthetic peptides containing residues 106-126 (CR) located in the central domain (CD, 95-133) of PrP(C) are neurotoxic. The central domain comprises two chemically distinct subdomains, the charge cluster (CC, 95-110) and a hydrophobic region (HR, 112-133). The aim of the present study was to establish the individual cytotoxicity of CC, HR and CD. Our results show that only the CD peptide is neurotoxic. Biochemical, Transmission Electron Microscopy and Atomic Force Microscopy experiments demonstrated that the CD peptide is able to activate caspase-3 and disrupt the cell membrane, leading to cell death.

Protein-protein interaction antagonists as novel inhibitors of non-canonical polyubiquitylation.

BACKGROUND: Several pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-kappaB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev. METHODOLOGY/PRINCIPAL FINDINGS: By applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-kappaB by TNF-alpha and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells. CONCLUSIONS/SIGNIFICANCE: This is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications.

Preparation of de novo globular proteins based on proline dendrimers.

A synthetic method for the preparation of protein-like globular dendrimers derived from a combination of proline, glycine and imidazolidin ring as branching unit is described. The methodology allows the synthesis of novel peptide dendrimers up to fourth generation. Dendrimers were synthesized by a convergent solid-phase peptide synthesis approach. The conformational properties of branched polyproline peptides and proline dendrimers were studied by CD experiments. CD data suggest conformational plasticity of branched peptides for PPI and PPII, and a stable well-defined secondary structure of proline dendrimers for PPII.

Synthesis and screening of a small library of proline-based biodendrimers for use as delivery agents.

A small library of defined peptide dendrimers based on polyproline sequences was designed to demonstrate the feasibility of generating a new type of polymeric agent for therapeutic use. Structural modifications to dendrimer surfaces further enriched the diversity of the library. Data show that the prolinerich dendrimers can be internalized in human epithelial (HeLa) cells, demonstrating the importance of the dendrimeric motif. The promising results described herein suggest that controlled modification of the dendrimer surface should eventually yield proline dendrimers with therapeutic potential.

Solid-phase synthesis of second-generation polyproline dendrimers.

Second-generation dendrimers have been prepared on solid phase by successive additions of branched polyproline building blocks starting from two different branching units anchored to the solid support. The preparation of Pro-rich building blocks was carried out by stepwise solid-phase synthesis and their iterative addition was performed by a convergent approach, also using solid-phase synthesis. cis-4-Amino-L-proline and imidazolidine-2-carboxylic acid were used as branching units due to their structural resemblance to proline. The optimized strategy allowed the target compounds to be obtained with high purities without the need for purification steps.

Peptide dendrimers based on polyproline helices.

We present a new family of peptide dendrimers based on polyproline helices and cis-4-amino-L-proline as a branching unit. Dendrimers were synthesized by a convergent solid-phase peptide synthesis approach. The conformational transition between polyproline type I helix and polyproline type II helix was observed by circular dichroism in branched polyproline building blocks with more than 14 proline residues and in the resulting dendrimers. Both linear and dendritic polyprolines were found to be actively internalized by rat kidney cells. Preliminary results show that the antibiotic ciprofloxacin form complexes with branched polyproline chains in 99.5% propanol.