Biomedical subjects

Olivier Seksek

Publications and source records attributed to Olivier Seksek.

5 recordsLinked to original sources

PubMed2006

Secondary conformation of short lysine- and leucine-rich peptides assessed by optical spectroscopies: effect of chain length, concentration, solvent, and time.

Solution secondary structures of three synthetic cationic peptides, currently used in antisense oligonucleotide delivery into living cells, have been analyzed by means of circular dichroism (CD) and Raman scattering in different buffers as a function of concentration and time. All three peptides are of minimalist conception, i.e., formed by only two types of amino acids (leucine: L and lysine: K). Two of these peptides contain 15 aminoacids: N(ter)- KLLKLLLKLLLKLLK (L(10)K(5)), N(ter)-KLKLKLKLKLKLKLK (L(7)K(8)), and the third one has only 9 residues: N(ter)-KLKLKLKLK (L(4)K(5)). The conformational behavior of the 15-mers in pure water differs considerably one from another. Although both of them are initially disordered in the 50-350 microM range, L(10)K(5) gradually undergoes a disordered to alpha-helix transition for molecular concentrations above 100 microM. In all other solvents used, L(10)K(5) adopts a stable alpha-helical conformation. In methanol and methanol/Tris mixture, nonnative alpha-helices can be induced in both KL-alternating peptides, i.e., L(7)K(8) and L(4)K(5). However, in major cases and with a time delay depending on peptide concentration, beta-like structures can be gradually formed in both solutions. In PBS and methanol/PBS mixture, the tendency for L(7)K(8) and L(4)K(5) is to form structures belonging to beta-family. A discussion has been undertaken on the effect of counterions as well as their nature in the stabilization of ordered structures in both KL-alternating peptides.

Amino Acid Sequence↗

PubMed2004

The role of structural factors in the kinetics of cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines: implications for overcoming multidrug resistance towards leukaemia K562/DOX cells.

The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer. Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient. Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs). The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line. The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells. It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC). The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake. In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein. Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR.

Acridines↗

PubMed2004

Complex formation and vectorization of a phosphorothioate oligonucleotide with an amphipathic leucine- and lysine-rich peptide: study at molecular and cellular levels.

Optical spectroscopic techniques such as CD, Raman scattering, and fluorescence imaging allowed us to analyze the complex formation and vectorization of a single-stranded 20-mer phosphorothioate oligodeoxynucleotide with a 15-mer amphipathic peptide at molecular and cellular levels. Different solvent mixtures (methanol and water) and molecular ratios of peptide/oligodeoxynucleotide complexes were tested in order to overcome the problems related to solubility. Optimal conditions for both spectroscopic and cellular experiments were obtained with the molecular ratio peptide/oligodeoxynucleotide equal to 21:4, corresponding to a 7:5 ratio for their respective +/- charge ratio. At the molecular level, CD and Raman spectra were consistent with a alpha-helix conformation of the peptide in water or in a methanol-water mixture. The presence of methanol increased considerably the solubility of the peptide without altering its alpha-helix conformation, as evidenced by CD and Raman spectroscopies. UV absorption melting profile of the oligodeoxynucleotide gave rise to a flat melting profile, corresponding to its random structure in solution. Raman spectra of oligodeoxynucleotide/peptide complexes could only be studied in methanol/water mixture solutions. Drastic changes observed in Raman spectra have undoubtedly shown: (a) the perturbation occurred in the peptide secondary structure, and (b) possible interaction between the lysine residues of the peptide and the oligodeoxynucleotide. At the cellular level, the complex was prepared in a mixture of 10% methanol and 90% cell medium. Cellular uptake in optimal conditions for the oligodeoxynucleotide delivery with low cytotoxicity was controlled by fluorescence imaging allowing to specifically locate the compacted oligonucleotide labeled with fluorescein at its 5'-terminus with the peptide into human glioma cells after 1 h of incubation at 37 degrees C.

Biological Transport↗

PubMed2004

Delivery agents for oligonucleotides.

This chapter provides a basic overview of most of the oligonucleotide delivery systems available for an in vitro use. Two major classes are described: systems that act through an endocytosis process (e.g., lipid-based vectors, nanoparticles, and polycations) and systems that by-pass this endocytosis process (e.g., peptides and pore-forming agents). Each technique is briefly described to allow a critical choice of the best delivery systems suitable for specific purposes in cultured cells.

Animals↗

PubMedDate not supplied

Intracellular uptake of modified oligonucleotide studied by two fluorescence techniques.

Interaction, i.e., cellular uptake and intracellular distribution, of synthetic modified antisense oligonucleotide with the B16 melanoma cell line was studied using cationic polyene antibiotic, amphotericin B 3-dimethylaminopropyl amide, as a carrier vector. The antisense oligonucleotide--dT(15) oligomer analogue containing isopolar, nonisosteric, phosphonate-based internucleotide linkages 3'-O-P-CH(2)-O-5'--was labeled with fluorescent tetramethylrhodamine marker. The oligonucleotide itinerancy across the cell membrane and its distribution inside the cell was visualized using fluorescence microimaging. During the first several hours a strong preference staining of the cell nucleus was found. Fluorescence lifetime measurements from the intracellular environment (confocal laser microspectrofluorimeter, frequency domain phase/modulation technique in 1 to 200 MHz frequency region) yielded two spectral components of 4.9 and 1.4 ns lifetime, respectively. While the former component correlates with the previously characterized effect of the fluorophore binding to biomolecular targets in membranes and/or cytoplasm, the latter component is newly observed and its possible origin is discussed.

Amphotericin B↗