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Synthesis, Biological Evaluation and Insights into the Mode of Action of Quinoxaline Containing Peptides

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Universitat de Barcelona. Facultat de Química

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[eng] Triostins are members of the quinoxaline antibiotic family, together with the "prototypical bisintercalator" echinomycin. These molecules display interesting antitumour activities as they inhibit transcription through insertion of their two planar quinoxaline rings into the DNA double helix, spanning two base pairs, while the peptide backbone is placed in the minor groove. The main objective of this Doctoral Thesis has been the design, synthesis and characterization of novel compounds with antitumoral activity, taking as basis the chemical structure of the natural bisintercalator triostin A. Synthetically challenging peptides bearing consecutive N-methyl and beta-branched amino acids were obtained after careful selection of a suitable coupling system for the solid-phase synthesis of the peptidic scaffolds. The introduction of the heterocycles and side-chains deprotection were carried out in solution as final stages. At the end, a small library of twelve peptides, fifteen quinoxaline-containing peptides and three fluorescent versions were obtained in excellent purities and employed in further biological evaluations. The synthesized compounds were evaluated for their antitumoral activity using the MTT assay, performing preliminar determinations of the detection limits of the assay, the lenght of the exponential phase of the cell lines tested and their doubling times. The most active compound was RZ2, with low micromolar cytotoxic activities, being in all cases more active than the natural parent compound triostin A, and with better cytotoxic activity than the commercial drug doxorubicin against cervical and breast adenocarcinoma cells. Unfortunatelly, compound RZ2 was also cytotoxic against non-tumor endotelial cells. However, this same compound resulted also active against the parasite Plasmodium falciparum. The stability of RZ2 in human serum and in presence of two overexpressed tumor proteases, MMP2 and cathepsin B, was studied. A little percentage of degradation was observed from 24 hours, with a maximum degradation of 30% in presence of serum at 48 hours. These data indicate that the peptide is advantageous in terms of proteolytic stability in vivo. Moreover, RZ2 is not hemolytic ex vivo, pointing out the great potential of this compound in antiplasmodial treatment without nocive effects on human erythrocytes. Furthermore, a liposomal formulation of compound RZ2 was prepared using egg phosphatidylcholine and cholesterol so as to obtain small unilamellar vesicles and improve the solubility of the quinoxaline-containing peptide. In regard of the mode of action of the synthesized compounds, it was demonstrated through circular dichroism, band-shift and DNase I footprinting experiments that there is no interaction with the genetic material. Molecular dynamics simulations showed that the analogues do adopt the predicted antiparallel beta-sheet conformation, but the separation between the quinoxaline moieties is too little to allow the sandwitching of two DNA base pairs. Non-synchronized HeLa cells cultures treated with compound RZ2 resulted in S phase-arrest. PI permeability assays coupled to annexin V labeling in HeLa cells showed increased PI/annexin V labeling in response to RZ2 in a time-dependant manner. Induction of apoptosis was confirmed by an increase in the sub-G1 DNA fragmentation in response to the compound over time. Furthermore, cells treated with RZ2 for 48 hours showed increase in active caspase 3 and poly(ADP-ribose) polymerase levels, confirming that HeLa cells die as a result of apoptosis when exposed to RZ2. Internalization into the cytosol was studied using a fluorescent version of RZ2 that displayed similar cytotoxicity. It was observed that the compound accumulates in acidic compartments and may inhibit the autophagic responsed triggering apoptosis. Changes in gene expression before the apoptotic pathway activation were studied using a gene expression microarray. Results suggest that RZ2 induces metabolic stress in HeLa cells.

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Synthesis, Biological Evaluation and Insights into the Mode of Action of Quinoxaline Containing Peptides