Immunoproteasom is a highly effective protease complex which is predominantly found in cells of the immune system, where it participates in antigen presentation, regulation of cytokine secretion, differentiation and proliferation of lymphocytes T. In non-hematopoietic cells its expression can be induced under the influence of proinflammatory cytokines or oxidative stress, where all three catalytically active β subunits of constitutive proteasome (β1, β2 in β5) are replaced by the corresponding homologous subunits of the immunoproteasome (β1i, β2i in β5i). Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases and various types of cancer. Therefore, immunoproteasome inhibition offers an attractive therapeutic approach in the treatment of these diseases.
In the course of our research, ten new compounds based on electrophilic oxathiazolone were evaluated as potential inhibitors of immunoproteasome. With methods of enzyme kinetics, we firstly determined IC50 values for the ten compounds on the β5i subunit. The values were in the range of 0.10-0.43 μM, with the lowest IC50 values for compounds 55, 172 and 176. Furthermore, we tested the selectivity of all the inhibitors against other catalytically active subunits of immunoproteasome (β1i, β2i) and proteasome (β1 , β2, β5). We found that at 10 μM concentration, the residual activity of all other subunits was above 60% for coumpound 55, 57 and 176. Based on this, we can conclude that these compounds selectively inhibit the β5i subunit of the immunoproteasome.
Compounds 55 and 172 had the lowest IC50 for the β5i subunit and at the same time exerted high selectivity towards other subunits, thus decided for further evaluation. In the first part, we checked whether the compounds retain their inhibitory properties in heterogeneous protein systems. For this purpose, cell lysates from THP-1 and HeLa cells were prepared. In cell lysates the IC50 values increased by about 10-fold relative to the isolated β5i subunit, suggesting that in complex systems the compounds bind to or inhibit other proteins. Further, we addressed the properties of cell permeability of the selected compounds in in vitro conditions. We demonstrate that the residual activity at 25 μM concentration was 65% and 74% for the compound 55 and 172 respectively. This indicates the poor permeability of the compounds via the intact cell membrane and the need for structure optimization.
In the last segment of our work, we were interested whether the compounds possess any immunomodulatory effects on peripheral mononuclear cells (PBMCs) of healthy individuals. PBMCs were activated with lipopolysaccharide and the potential effect of the two compounds on secretion of inflammatory cytokines (IL-1β, TNFα, IL-8, IL-6, IL-10, and IL-12p70) was determined. We found that the statistical reduction in IL-6 and IL-10 was caused only by compound 172. A similar trend was also observed for compound 55, however it was not statistically significant.
To conclude, the compounds show great potential as selective inhibitors of the β5i subunit of immunoproteasom, but further structural optimization would be required to improve permeability over the cell membrane.
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