Synthesis, Structural and Biological Analysis of Organized Biomimetic Systems
Creators: name: Lubomir, Vezenkov
Publisher: name: Università degli Studi di Napoli Federico II
Year: 2010

description: As a part of a program for foldamer design two β-turn mimetics (3S)-amino-5-(carboxylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one or DBT and 2-aminomethyl-phenyl-acetic acid or AMPA were selected as frameworks from a molecular modeling study for their suitability to adopt helical structure.  By standard solid phase peptide synthesis (SPPS) we synthesized DBT and AMPA oligomers of different lengths and modifications were introduced at their N-terminus. Our first task was to perform structural analysis of the oligomers by NMR and X-Ray. Numerous NOE interactions in the DBT pentamer and hexamer molecules were detected by NMR 2D NOESY experiments. These data strongly suggest the organization of these DBT oligomers.  In a parallel study we hypothesized that short oligomers constructed by DBT or AMPA frameworks could translocate the cellular membrane and could be used as new cell penetrating non-peptides - CPNP.  Even though these compounds are not charged as most cell penetrating peptides (CPP) or CPNP, we considered that by virtue of their aromaticity, hydrophobicity and their well-organized structure they could have a non-specific interaction with the lipid bilayer and thus be internalized into the cell.  At first the cellular uptake of the (DBT)2-4 oligomers in MDA-MB-231 breast cancer cells was analyzed by fluorescence emission measurement and compared to the potent and well-studied CPP octa-arginine (Arg)8 as a positive control and carboxyfluorescein as a negative control. The highest intracellular fluorescence intensity was found for (DBT)4 with a drastic decrease (&gt;4-times) for (DBT)3 and (DBT)2 oligomers. Thus, the cellular uptake appeared length-dependent with an increase of the internalization with the oligomer size. Moreover, the amount of (DBT)4 that was internalized was more significant than that of (Arg)8 despite the fact that it is uncharged. By confocal microscopy we determined that (DBT)4 is mainly localized in the endosomes after 3 hours of incubation and in the lysosomes after 16 hours of incubation. Similar confocal microscopy data was obtained with the (AMPA)4  oligomer. Altogether, these results indicate the ability of these oligomers to target the endolysosomal pathway which could be useful for drug delivery in the treatment of lysosomal storage diseases, Alzheimer’s disease, and cancer. 
 
We also developed a new general methodology for the determination of the intracellular concentration of CPP/CPNP based on MALDI-TOF mass spectrometry (MS) which does not require any purification or separation steps. We studied the internalization of CPP/CPNP compounds by using an UV light-absorbing tag alpha-cyano-4-hydroxycinnamic acid (HCCA) and preparing the samples in a neutral matrix such as alpha-cyano-4-hydroxycinnamic methyl ester (HCCE). This combination (HCCA tag and HCCE matrix) enabled us to discriminate MS signals induced by peptides of interest that were present in low concentration from those of unlabelled more abundant peptides. By addition of a precise amount of deuterated-HCCA-tagged CPP/CPNP prior the MALDI TOF MS experiment, the internalized CPP/CPNP could be quantified on the basis of the ratio between the [M+H]+ peaks of the deuterated and nondeuterated HCCA-tagged CPP. 
 
In order to prove that our new CPNP could translocate biologically relevant cargos we connected them to the powerful inhibitor of the Cahtepsin D enzyme - pepstatine. Such compounds could be a potential anti-cancer drug because the Cathepsin D is implicated in the cancer development and proliferation. The non vectorized pepstatine is inactive in vitro because of its inhability to cross the lipid bilayer and to reach the endosomes where the Cathepsin D mitogenic activity is supposed to be very important. Pepstatine vectorized by the (AMPA)4 CPNP showed considerable toxicity in vitro probably via the inhibition of the Cathepsin D enzyme. Tests for anti-cancer activity on mice will be performed in the near future.