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Développement d’une sonde de photoaffinité pour la détection sensible de formes actives de Métalloprotéases Matricielles dans des systèmes biologiques complexes

Abstract : A new activity-based probe able to covalently modify the active site of proteases belonging to the matrix metalloprotease family (MMPs) has been developed in this thesis project. The probe was shown to behave as potent inhibitor of several MMPs, with nanomolar Ki values. This probe was also able to modify specifically only the free active site of MMPs, with particular high yields of cross-linking varying from 50 % to 11 %, depending of the MMPs tested. Using radioactivity as means of detection, this probe was able to detect active form of MMPs with a threshold of 1 femtomole. Applied to the study of bronchoalvelolar fluids (BAL) from mice exposed to nanoparticles by a lung aspiration protocol, this probe revealed the presence of the catalytic domain of MMP-12 under its active form, but not in control animals. When used to detect active form of MMPs from extracts obtained from human arteries of patient suffering from atherosclerosis, the probe was not able to detect such MMP active forms. Despite this negative result, the detection of active form of MMP in pathological fluid like BAL has never been reported before this work. Having validated this novel MMP activity-based probe, it will be possible to use it now for detecting MMPs from other pathological fluids or tissues extracts in which MMPs can be good markers of the pathology.
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Catherine Nury. Développement d’une sonde de photoaffinité pour la détection sensible de formes actives de Métalloprotéases Matricielles dans des systèmes biologiques complexes. Médecine humaine et pathologie. Université René Descartes - Paris V, 2012. Français. ⟨NNT : 2012PA05P629⟩. ⟨tel-00868790⟩

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