Phenoxypropanolamine derivatives as selective inhibitors of the 20S proteasome ß1 and ß5 subunits.

New series of thiophene-containing phenoxypropanolamines were synthesized and evaluated for their potency to inhibit the three proteolytic activities of the mammalian 20S proteasome. Noticeable inhibition of both ChT-L and PA activities was obtained with three compounds: one with unsubstituted phenoxypropanolamine group (7) and the two others with a p-Cl-substituted group (4 and 9). For three other compounds (3, 8 and 10), ChT-L activity alone was significantly inhibited. In silico docking performed on the ß5 and ß1 subunits bearing the respective ChT-L and PA catalytic sites showed features common to poses associated with active compounds. These features may constitute a selectivity criterion for structure-guided inhibitor design.

Bisbenzimidazole derivatives as potent inhibitors of the trypsin-like sites of the immunoproteasome core particle.

In this study, a monomeric (MB) and a dimeric (DB) bisbenzimidazoles were identified as novel proteasome inhibitors of the trypsin-like activity located on ß2c sites of the constitutive 20S proteasome (IC50 values at 2-4 µM range). Remarkably, they were further shown to be 100- and 200-fold more potent inhibitors of the immunoproteasome trypsin-like activity (ß2i sites, IC50=24 nM) than of the homologous constitutive activity. Molecular models of inhibitor/enzyme complexes in the two types of trypsin-like sites and corresponding computed binding energy values corroborated kinetic data. Different binding modes were suggested for MB and DB to the ß2c and ß2i trypsic sites. Each pointed to better contacts of the ligand inside the ß2i active site than for ß2c site. MB and DB represent the first selective inhibitors of the immunoproteasome trypsin-like activity described to date and can be considered as prototypes for inhibiting this activity.

New C(4)- and C(1)-derivatives of furo[3,4-c]pyridine-3-ones and related compounds: evidence for site-specific inhibition of the constitutive proteasome and its immunoisoform.

A set of 18 new C(4) and C(1) derivatives of nor-cerpegin (1,1-dimethyl furo[3,4-c]pyridine-3-one), 6 model compounds (γ- and δ-lactones) and 20 furo- or thieno[2,3-d]-pyrimidine-4-one related compounds were designed and synthesized. Each compound was assayed for inhibition of CT-L, T-L and PA proteolytic activities of 20S constitutive proteasome (c20S). Most performant compounds were also assayed on 20S immunoproteasome (i20S). Compound 10 with a benzylamino group at C(4) and dimethylated at C(1) of the furopyridine ring was the most efficient PA site-specific inhibitor of the c20S (IC50(cPA) of 600nM) without noticeable inhibition of the i20S PA site (iPA). In silico docking assays for 10 at the iPA catalytic site revealed the absence of poses normally observed for this compound and related ones at the constitutive PA site (cPA). The thieno[2,3-d]pyrimidine-4-one 40 was T-L site-specific with a mild inhibition of both c20S and i20S in vitro (IC50(cT-L) of 9.9µM and IC50(iT-L) of 6.7µM). In silico docking assays of 40 at T-L sites of c20S and i20S revealed almost identical first rank poses in the two types of sites with no possibility left for nucleophilic attack by Thr1 as observed for the fused furopyridine-3-one 10.

C1 and N5 derivatives of cerpegin: synthesis of a new series based on structure-activity relationships to optimize their inhibitory effect on 20S proteasome.

Thirty-two new derivatives of cerpegin (1,1,5-trimethylfuro[3,4-c]pyridine-3,4-dione) were designed and synthesized in high yield by a new method, combining several C(1) and N(5) substituents. All compounds were tested for their inhibitory effect on the CT-L, T-L and PA proteolytic activities of a purified mammalian 20S proteasome. Only one molecule inhibited both CT-L and PA activities. Sixteen molecules specifically inhibited PA at the micromolar range, out of which fourteen had IC50 values around 5 µM and two had IC50 values closer to 2 µM. Except in one case, neither calpain I nor cathepsin B was inhibited. In silico docking suggests a unique mode of binding of the most efficient compounds to the ß1 catalytic site (PA activity) in relation to the chemical nature of C(1) substituents.

A new series of N5 derivatives of the 1,1,5-trimethyl furo[3,4-c]pyridine-3,4-dione (cerpegin) selectively inhibits the post-acid activity of mammalian 20S proteasomes.

A large set of N(5)-derivatives of cerpegin (1,1,5-trimethyl furo[3,4-c]pyridine-3,4-dione) was designed and synthesized in high yields by a simple and handy method using various primary amines for a pyridine cycle synthesis. The effects of 29 derivatives on the three types of catalytic sites of purified mammalian 20S proteasomes (CT-L, T-L and PA) were measured. Most of the new compounds specifically inhibited the PA activity, in the micromolar range. Docking experiments support these results. Moreover, neither calpain I nor cathepsin B were inhibited.

Novel organic proteasome inhibitors identified by virtual and in vitro screening.

Proteasome inhibition is a promising strategy for treating cancers. Herein, we report the discovery of novel drug-like inhibitors of mammalian proteasome 20S using a multistep structure-based virtual ligand screening strategy. Sulfone- or piperazine-containing hits essentially belong to the under-represented class of noncovalent and nonpeptidic proteasome inhibitors. Several of our compounds act in the micromolar range and are cytotoxic on human tumoral cell lines. Optimization of these molecules could lead to better anticancer therapy.

Novel fluorinated pseudopeptides as proteasome inhibitors.

We have designed novel small inhibitors of rabbit 20S proteasome using a trifluoromethyl-beta-hydrazino acid scaffold. Structural variations influenced their inhibition of the three types of active sites. Proteasome inhibition at the micromolar level was selective, calpain I and cathepsin B were not inhibited.