Christian Dubiella

20S proteasomes represent the central proteolytic machineries which break down intracellular proteins. Based on their participation in cell proliferation, survival and apoptosis, proteasomes are targets in cancer therapy. Here, clinically applied proteasome inhibitors induce cell death by blocking both the constitutive and the immunoproteasome. The resulting cytotoxicity provides the rationale for the treatment of blood cancers, however, it limits the clinical utility of unselective proteasome blockers to chemotherapy.

In contrast, the selective inhibition of the catalytic subunit β5i of the immunoproteasome is less cytotoxic and has the potential to modulate immune disorders as shown in mouse models of several autoimmune diseases and chronic inflammations. In addition, selective immunoproteasome blockers as research tools play an important role in the investigation of the immunoproteasome’s involvement in antigen presentation and regulation of cytokine, both processes of the immune system which are still not fully understood.

Motivated by this, I set out in my doctoral research to develop immunoproteasome inhibitors that preferentially target the immunoproteasome with high specificity. I followed an interdisciplinary approach which combined organic synthesis of candidate inhibitors, their biochemical evaluation and ultimately structural characterization with X-Ray crystallography. This holistic approach enabled me to characterize three new classes of selective immunoproteasome inhibitors:

1) peptidic α-chloroacetamides as the first immunoproteasome inhibitors that act independently from the active site by targeting a cysteine residue which was identified as new molecular target. These nanomolar binders exhibited low cytotoxicity and structure-based optimization led to over 150-fold selectivity for the immunoproteasome versus the proteasome. Investigations on immune cells showed that these binders indeed can suppress the production of cytokines, thereby displaying anti-inflammatory properties (for further reading, please see Dubiella et al., Angew. Chem. Int. Ed. 2015, 54 (52), 15888-91).

2) peptidic sulfonyl fluorides whose mode of action cause an intramolecular crosslinking of the active site, which was uncovered by time-resolved snapshots of the distinct reaction intermediates using X-Ray crystallographic analysis. For the first time, a pharmacophore was shown to enhance immunoproteasome selectivity (28-fold selectivity; for further reading, please see Dubiella et al., Angew. Chem. Int. Ed. 2014, 53 (44), 11969-73).

3) peptidic sulfonate esters as tunable probes for the constitutive and immunoproteasome. The inhibitory strength of these compounds can be altered by the choice of the leaving group. We found a clear correlation between the pK_a value of the corresponding acid of the leaving group and the potency of the inhibitors. Exchange of the leaving group with a fluorophore (DiFMU) with a favorable pK_a value allowed us to monitor the inhibition of proteasomes in real-time. Therefore, this study gave insights into the kinetics of proteasomal inhibition and provides tools to directly quantify active proteasomes in solution with high sensitivity (for further reading, please see Dubiella et al., Angew. Chem. Int. Ed. 2016, in print.)