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Content archived on 2024-05-29

Novel molecular tools for the analysis of the cell membrane proteome and interactome

Final Activity Report Summary - MOLTOOL / INTERACTOM (novel molecular tools for the analysis of the cell membrane proteome and interactome)

The project aimed at the development of new chemical tools to allow the creation and analysis of proteome and interactome subsets. For this purpose, small molecule ligands of the proteins of interest were chemically modified to allow their immobilisation on a solid support like SPR chip or affinity chromatography material. Focus was placed on two protein families, Matrix metalloproteinases (MMP's) and integrins, as they are targets in tumour therapy.

Marimastat is a known and efficient inhibitor of MMP's which present a broad activity among this family of proteins. This lack of selectivity make it an ideal candidate for derivatisation. Based on this conclusion, two new probes, which contain the marimastat moiety derivatised by a spacer to allow their immobilisation on a solid support, were designed and synthesised.

Several bioassays were established concerning these probes. The inhibitory activities of the two marimastat derivatives were tested towards MMP's, and both present a good inhibitory activity in the nanomolar range, comparable to the values obtained for the parent compound. However, the derivative containing the longer spacer present a better activity than the shorter one, so it seems that the spacer should present a minimal length to not disturb the binding of the ligand with the targeted proteins.

Affinity chromatography of complex systems like placenta extract have then been performed with these immobilised derivatives using a tailor-made affinity column. Under optimised elution conditions, it has been possible, for the first time, to bind the targeted proteins together with their interaction partners to the immobilised marimastat derivative, and to release them subsequently to create a proteome and interactome subset. Analysis of the proteins of this subset has revealed some proteins that were expected, like MMP's and integrins, which are known to interact with each other, but also some others like ADAM10, cytochrome C, fibronectin, laminin, actin... Proteins like ADAM10 and cytochrome C seem to interact directly with the immobilised marimastat derivative, but for the other proteins, it is less clear, and they may correspond to proteins interacting with the targeted ones.

At the same time, another marimastat derivative containing a photoreactive moiety and a fluorophor as reporter group has also been synthesised and tested. It presents a good inhibitory activity towards several MMP's, which is a major precondition for a successful application of the concept to the family of MMP's. Studies have been realised with this inhibitor to optimise the conditions of the photoreaction creating a covalent bond between the organic compound and the proteins. This kind of probe can be used to differentiate between the proteins directly targeted by marimastat derivatives and interaction partners.

Concerning the integrin part of the project, the triad Arg-Gly-Asp (RGD) is known to be a recognition sequence found in the proteins which interact with integrins. Consequently, peptides which contain this sequence should be good ligands for integrins. In this context, three cyclic peptides containing this RGD sequence have been synthesised; one of them was derivatised with a spacer to allow its immobilisation on a solid support. The inhibitory activities of the three compounds have been tested towards integrins alpha5 beta1 and alphav beta3, and have been found to present a good inhibitory activity in the low nanomolar range. It seems that the derivatisation with a spacer does not affect the inhibitory activity of the peptide.

Surface plasmon resonance (SPR) studies have then been realised on placenta extract containing integrins, to determine the optimum conditions for affinity chromatography expemiments. They have shown that it is possible to bind proteins from the placenta extract to the immobilised peptide, and to release them subsequently. These results show that affinity chromatography experiments, using the immobilised peptide, should be promising for the analysis of integrin proteome and interactome.