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

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 (<i>K</i>_i = 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

Carbonylhydrazide-Based Molecular Tongs Inhibit Wild-Type and Mutated HIV‑1 Protease Dimerization

We have designed and synthesized new molecular tongs based on a rigid naphthalene scaffold and evaluated their antidimer activity on HIV-1 protease (PR). We inserted carbonylhydrazide and oligohydrazide (azatide) fragments into their peptidomimetic arms to reduce hydrophobicity and increase metabolic stability. These fragments are designed to disrupt the protein–protein interactions by reproducing the hydrogen bond pattern found in the antiparallel β-sheet formed between the <i>N</i>- and <i>C</i>-ends of the two monomers in the native PR. Kinetic analyses and fluorescent probe binding studies showed that several molecular tongs can inhibit PR dimerization. The best nonpeptidic molecular tongs to date were obtained with an inhibition constant <i>K</i>_id of 50 nM for PR and 80 nM for the multimutated protease ANAM-11. The PR inhibition was selective, the aspartic proteases renin and pepsin were not inhibited