Joëlle Vidal

Proteasome inhibitors: recent advances and new perspectives in medicinal chemistry

The search for proteasome inhibitors began fifteen years ago. These inhibitors proved to be powerful tools for investigating many important cellular processes regulated by the ubiquitin-proteasome pathway. Targeting the proteasome pathway can also lead to new treatments for disorders like cancer, muscular dystrophies, inflammation and immune diseases. This is already true for cancer; the FDA approved bortezomib, a potent proteasome inhibitor, for treating multiple myeloma in 2003, and mantle cell lymphoma in 2006. The chemical structures identified in some of the early proteasome inhibitors have led to the development of new anti-cancer drugs (CEP-18770, Carfilzomib, NPI-0052). All these molecules are covalent bonding inhibitors that react with the catalytic Thr1-O(gamma) of the three types of active site. This review covers recent developments in medicinal chemistry of natural and synthetic proteasome inhibitors. Advances in non-covalent inhibitors that have no reactive group will be highlighted as they should minimize side-effects. New structures and new modes of action have been recently identified that open the door to new drug candidates for treating a range of diseases.

20S Proteasome Inhibition: Designing Noncovalent Linear Peptide Mimics of the Natural Product TMC-95A

The 20S proteasome maintains homeostasis and regulates important intracellular processes by subjecting most intracellular proteins to endoproteolytic cleavage. This complex hydrolysis machinery has received considerable attention for the treatment of many diseases, including cancer. We report a new set of noncovalent peptide mimics based on TMC-95A and our rational approach to inhibitor optimization using both crystallographic and kinetic studies.

Dimerized Linear Mimics of a Natural Cyclopeptide (TMC-95A) Are Potent Noncovalent Inhibitors of the Eukaryotic 20S Proteasome

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors used in cancer therapy. Starting from a noncovalent linear mimic of TMC-95A, a series of dimerized inhibitors using polyaminohexanoic acid spacers has been designed and optimized to target simultaneously two of the six active sites of the eukaryotic 20S proteasome. The homodimerized compounds actively inhibited chymotrypsin-like (Ki = 6-11 nM) and trypsin-like activities, whereas postacid activity was poorly modified. The noncovalent binding mode was ascertained by X-ray crystallography of the inhibitors complexed with the yeast 20S proteasome. The inhibition of proteasomal activities in human cells was evaluated. The use of the multivalency inhibitor concept has produced highly efficient and selective noncovalent compounds (no inhibition of calpain and cathepsin) that have potential therapeutic advantages compared to covalent binders such as bortezomib and carfilzomib.

Noncovalent inhibition of 20S proteasome by pegylated dimerized inhibitors

We exploited the concept of polyvalent interactions to produce highly selective and efficient inhibitors of eukaryotic proteasome. This multicatalytic protease with the unique topography of its 6 active sites has emerged as a promising target to treat cancer with the use of the covalent inhibitor bortezomib. We used our reference noncovalent inhibitor, a selective TMC-95A tripeptide linear mimic, to design dimeric noncovalent proteasome inhibitors that target two active sites simultaneously. We synthesized pegylated monomer and dimers of the reference inhibitor and evaluated their capacity to inhibit a mammalian 20S proteasome. The inhibitory power of the dimers depended on the average length of their spacer. Lineweaver-Burk double-reciprocal plots indicated competitive inhibition. The best dimer inhibited CT-L activity 800-times more efficiently than the reference inhibitor.

Blockade of the malignant phenotype by β -subunit selective noncovalent inhibition of immuno- and constitutive proteasomes

A structure-based virtual screening of over 400,000 small molecules against the constitutive proteasome activity followed by in vitro assays led to the discovery of a family of proteasome inhibitors with a sulfonyl piperazine scaffold. Some members of this family of small non-peptidic inhibitors were found to act selectively on the β2 trypsin-like catalytic site with a preference for the immunoproteasome β2i over the constitutive proteasome β2c, while some act on the β5 site and post-acid site β1 of both, the immunoproteasome and the constitutive proteasome. Anti-proliferative and anti-invasive effects on tumor cells were investigated and observed for two compounds. We report novel chemical inhibitors able to interfere with the three types of active centers of both, the immuno- and constitutive proteasomes. Identifying and analyzing a novel scaffold with decorations able to shift the binders’ active site selectivity is essential to design a future generation of proteasome inhibitors able to distinguish the immunoproteasome from the constitutive proteasome.

Noncovalent Fluorescent Probes of Human Immuno- and Constitutive Proteasomes

We report here the synthesis and biological evaluation of fluorescent probes functioning as inhibitors that noncovalently block human immuno- and constitutive proteasomes. These cell-penetrating linear analogues of the natural cyclopeptide TMC-95A were efficient on cells at the nanomolar level and assessed by confocal microscopy and flow cytometry. They may constitute an alternative to previously reported fluorescent probes that all bind covalently to proteasomes.