Abstract
Cathepsin D (CathD) is an overexpressed lysosomal protease secreted by several solid tumors. This enzyme is involved in tumor proliferation and metastasis, which makes it a promising target for cancer therapy. There exists a potent CathD inhibitor called Pepstatin, but it presents a too poor ability to cross the plasmic membrane in order to be active. To overcome this drawback, the development of cell penetrating vectors based on the oligomerization of constrained dipeptide mimetics has been undertaken in our lab. From this work, the bioconjugate JMV4463 has been developed. This bioconjugate is made of an AMPA4 (2-(aminomethyl)phenylacetic acid) vector, the pepstatin and a hydrophilic part which increases its solubility. This bioconjugate is able to penetrate the cells and has an antiproliferative effect on different cancer cell lines. Based on this work, synthesis of new potential cell penetrating vectors, oligomers of constrained dipeptide mimetics, was performed. Their internalization ability was established as their conjugate with pepstatin. We developed analogues of AMPA scaffold and (S)-2-(3-amino-4-oxo-3,4-dihydrobenzo[b][1,4]thiazépin-5(2H)-yl)acetic acid (DBT), which showed an important internalization ability. Thus, AMPA derivatives in indole, pyrrole and cyclohexane series were prepared. Furthermore, the synthesis of AMPA analogues having different substitutions on its aromatic part was performed, such as meta- and para- AMPA, an inferior homologue or 4,5-dimethoxy-, 4,5-dihydroxy- and 4-bromo-AMPA. An analogue of DBT in the benzodiazepin series has also been studied. All the corresponding vectors were able to internalize the pepstatin into the cells. Surprisingly, none of the conjugates showed an anti-proliferative activity, indicating the essential role of the AMPA vector. A structure/activity relationships study was performed for JMV4463. The obtained results confirmed that the AMPA vector has a unique mode of action that has not been identified yet. Furthermore, it has been demonstrated that it was possible to simplify the pepstatin structure by replacing one of its statin residues by a gamma alanine, which allows decreasing the production cost. Finally, an in vivo study on a xenografted mouse model showed a high anti-tumour potential for the JMV4463 conjugate.