S-2209, a Novel, Non-Boronic Proteasome Inhibitor, Induces Apoptosis and Cell Growth Arrest in Multiple Myeloma Cells.

Purpose: Multiple Myeloma (MM) is still an incurable disease. Patients become resistant to cytotoxic drugs and die of disease progression. Bortezomib is the first approved member of a new class of antineoplastic agents, the proteasome inhibitors. It has synergistic effects with genotoxic drugs and steroids in vitro and in vivo. However, single agent activity in humans is only moderate and specific toxicity (e.g. neurotoxicity) often limits its clinical use. Further proteasome inhibitors need to be developed to optimize this promising treatment option.

Methods: The new proteasome inhibitor S-2209 was characterized by several assays. Inhibition of the chymotryptic activity of the human 20S proteasome was determined with the in-vivo protease inhibition assay. Additionally, proteasome inhibition was determined in isolated PBMCs from S2209-pretreated wistar rats. Inhibition of NFκB activity was determined using a NFκB reporter gene assay. Cell growth rates of MM cells (OPM-2, U266, RPMI-8226 and NCI-H929) were measured with the WST-1 assay. Induction of apoptosis was shown by flow cytometry after staining with annexin-V-FITC and propidium iodide. Intracellular signal modulation was demonstrated by western blotting. Toxicity of the substance was tested in male wistar rats.

Results: The proteasome inhibition assay revealed an IC₅₀ at ∼220nM. The NFκB inhibition assay using an A549-NFκB-SEAP transfected cell line showed an EC₅₀ of 0.9μM. Upon incubation with S-2209, cell growth as well as cell proliferation in MM cell lines was significantly inhibited (IC₅₀ 100nM – 600nM). Furthermore, the incubation with S-2209 resulted in strong induction of apoptosis in all four MM cell lines even at nanomolar concentrations (IC₅₀ at ∼300nm) as well as primary cells. Western blotting revealed caspase-3 cleavage and upregulon of p53 and increased phosphorylation of IκB. No induction of apoptosis was detected in PBMCs from healthy humans. Despite the administration of 5, 10 or 15mg/kg/day in wistar rats, no toxicity with respect to body weight, hepatic enzymes (ALAT ASAT, ALP), creatinin or hemoglobin was seen. Proteasome inhibition in white blood cells isolated from the treated rats was higher in the S-2209 treated animals than in control animals treated with 0.1mg/kg/d bortezomib (89% vs. 70% respectively).

Conclusions: The proteasome inhibitor S-2209 inhibitis MM cell growth and induces apoptosis. This is accompanied by a strong inhibition of proteasome and of the NFκB activity. Because S-2209 shows a favourable toxicity profile in vivo, further clinical development of this promising drug is urgently needed.