Aminopeptidase-N (APN or CD13) [EC.3.4.11.2] is a highly glycosylated type II membrane-bound ectoenzyme that belongs to the M1 family of zinc-dependent aminopeptidases. The members of this family have a thermolysin-like catalytic domain with the consensus HEXXH-X18-E zinc-binding sequence and an exopeptidase motif, GXMEN, in the active site. APN/CD13 is a widespread enzyme, located in many tissues, organs and cells, whose multiple functions dependent on its location. It is overexpressed on the endothelial cells of angiogenic, but not normal, vasculature, as well as on numerous tumor cells. As it was demonstrated that APN plays a critical role in tumor cell angiogenesis and metastasis, this protein was identified as a potential target for cancer therapy. In this context, highly potent and selective non-peptidic APN inhibitors, with Ki values ranging from micro to nanomolar, were previously designed and synthesized in the laboratory. In vitro and in vivo efficacy of these novel amino-benzosuberone derivatives was tested. In parallel to these works, a new project was started a few years ago which consists to solve the 3D structure of mammalian APN. Co-crystallizations with amino-benzosuberone derivatives would determine the binding mode of these novel inhibitors. Nevertheless solving the structure of a membrane protein like mammalian APN still remains a challenge. Therefore several cloning and expression strategies for human APN production were developed.
