C-1 and N-5 derivatives of cerpegin: Synthesis of a new series based on structure-activity relationships to optimize their inhibitory effect on 20S proteasome

International audienceThirty-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 mu M and two had IC50 values closer to 2 mu M. 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 beta 1 catalytic site (PA activity) in relation to the chemical nature of C-1 substituents

G2-and early-M-specific expression of the NTCYC1 cyclin gene in Nicotiana tabacum cells

We have previously reported the isolation of a cDNA encoding a mitotic cyclin, NTCYC1, from a tobacco cell suspension library. Here we describe the expression patterns of NTCYC1 and pf Ntsuc1; a suc1 plant homologue, in synchronized tobacco cell suspensions. Furthermore, the expression pattern of this cyclin is compared to that of Ntcdc2-1, a Nicotiana tabacum homologue of cdc2. While no NTCYC1 transcript was detected in cells synchronized in the G1 and S phases, NTCYC1 expression was observed in late G2 and early M phases, disappearing in the G1' of a new cell cycle. On the other hand, Ntsuc1 and Ntcdc2-1 exhibited a constitutive expression during the cell cycle. A functional analysis performed by microinjecting NTCYC1 mRNA into immature Xenopus oocytes, indicates that NTCYC1 could participate in the control of the G2/M transition in plant cells. Subsequently NTCYC1 expression was used to assess the status of mesophyll cells in expanded leaves of N. tabacum. Depending on leaf position along the shoot axis, a large population of mesophyll cells appeared with a 4C DNA content, suggesting a G2 arrest. It was found that leaves with such a population also contained high levels of NTCYC1 transcripts. With respect to these results concerning a naturally occurring G2-arrested cell population, the regulation of NTCYC1 expression in planta is discussed

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

International audienceA 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 (gamma- and delta-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 (IC50cPA of 600 nM) 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 (IC50T-L of 9.9 lM and IC50T-L of 6.7 mu M). 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

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

International audienceIn this study, a monomeric (MB) and a dimeric (DB) bisbenzimidazoles were identified as novel proteasome inhibitors of the trypsin-like activity located on beta 2c sites of the constitutive 20S proteasome (IC50 values at 2-4 mu M range). Remarkably, they were further shown to be 100- and 200-fold more potent inhibitors of the immunoproteasome trypsin-like activity (beta 2i 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 beta 2c and beta 2i trypsic sites. Each pointed to better contacts of the ligand inside the beta 2i active site than for beta 2c 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

Novel Organic Proteasome Inhibitors Identified by Virtual and in Vitro Screening

International audienceProteasome 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