Partenaires et organisations internationales
(Anglais)
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AT, BE, CH, CS, CY, CZ, DE, ES, FI, FR, GR, HU, IT, LT, NL, NO, PL, PT, RO, SE, UK
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Résumé des résultats (Abstract)
(Anglais)
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Pyridoxal 5'-phosphate (PLP) is the functional cofactor of vitamin B6-dependent enzymes which play a central role in amine and amino acid metabolism and are potentially interesting targets for drugs intervening in processes that lead to pathological states. An obligatory step in all these reactions is the intermediary formation of an aldimine between PLP and the amino group of the substrate. Phosphopyridoxyl-amino acid adducts are a kind of transition state analogs of these enzymes and bind with very high affinity and specificity to their respective apoenzymes. The drawback of these compounds is that they are not taken up by cells due to the impermeability of cell membranes for them. Thus, we designed derivatives which we expected to be taken up by cells and converted within the cells to specific ligands of newly synthesized cellular vitamin B6-dependent apoenzymes. As a first target we have chosen ornithine decarboxylase (ODC), which is overexpressed in many tumor cells. The designed pyridoxyl-ornithine analog (POB) showed favorable interaction in the active site of hODC by molecular modeling methods. POB was synthesized, purified and tested for its inhibitory activity of cellular ODC and on cell growth. POB at 0.1 mM concentration inhibited the activity of serum induced newly synthesized ODC more than 90 % in glioma LN229 and COS7 cells. In correlation with the inhibition of ODC activity, we observed a time dependent inhibition of cell growth in all of the tested cell lines, such as myeloma, glioma, Jurkat and COS7, if cell number and DNA synthesis were measured (IC50 between 10 microM and 50 microM). We created and tested more transition-state mimics for human ornithine decarboxylase. Out of 23 synthesized compounds two more inhibitors have been found that also inhibit proliferation of glioma tumor cells with even better efficiency. Beside a hydrophobic side chain and a polyamine-like motif they feature a second mode of possible anti-proliferative action as they are inducing the activity of polyamine catabolic enzymes. Although these compounds are efficient inhibitors only for newly induced ODC in cells, they are much more effective in the proliferation-inhibiting potential of tumor cells than alpha-difluoromethylornithine, the well known irreversible ODC inhibitor. Additionally, we designed, synthesized and tested 9 potential membrane-permeable pyridoxyl-substrate conjugates as inhibitors for human HDC and modeled an active site of hHDC, which is compatible with the experimental data. The most potent inhibitory compound was a pyridoxyl-histidine methyl ester conjugate. It inhibited 60% of TPA-induced newly synthesized HDC in human HMC-1 cells at 200 µM and was also inhibitory in cell extracts.
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