Christian Dubiella

The concept of proteasome inhibition ranks among the latest achievements in the treatment of blood cancer and represents a promising strategy for modulating autoimmune diseases. In this study, we describe peptidic sulfonyl fluoride inhibitors that selectively block the catalytic β5 subunit of the immunoproteasome by inducing only marginal cytotoxic effects. Structural and mass spectrometric analyses revealed a novel reaction mechanism involving polarity inversion and irreversible crosslinking of the proteasomal active site. We thus identified the sulfonyl fluoride headgroup for the development and optimization of immunoproteasome selective compounds and their possible application in autoimmune disorders.

The 20S proteasome core particle (CP) is the proteolytically active key element of the ubiquitin proteasome system that directs the majority of intracellular protein degradation in eukaryotic cells. Over the past decade, the CP has emerged as an anticancer therapy target after approval of the first-in-class drug bortezomib (Velcade(®)) by the US Food and Drug Administration. However, bortezomib and all second-generation CP inhibitors that are currently explored in clinical phase studies react covalently and most often irreversibly with the proteolytic sites of the CP, hereby causing permanent CP blockage. Furthermore, reactive head groups result in unspecific binding to proteasomal active centers and in substantial enzymatic off-target activities that translate to severe side effects. Thus, reversible proteasome inhibitors might be a promising alternative, overcoming these drawbacks, but are challenging with respect to their urge for thorough enthalpic and entropic optimization. This review describes developments in the hitherto neglected field of reversible proteasome inhibitors focusing on insights gained from crystal structures, which provide valuable knowledge and strategies for future directions in drug development.

The ubiquitin-proteasome system (UPS) has been successfully targeted by both academia and the pharmaceutical industry for oncological and immunological applications. Typical proteasome inhibitors are based on a peptidic backbone endowed with an electrophilic C-terminus by which they react with the active proteolytic sites. Although the peptide moiety has attracted much attention in terms of subunit selectivity, the target specificity and biological stability of the compounds are largely determined by the reactive warheads. In this study, we have carried out a systematic investigation of described electrophiles by a combination of in vitro, in vivo, and structural methods in order to disclose the implications of altered functionality and chemical reactivity. Thereby, we were able to introduce and characterize the class of α-ketoamides as the most potent reversible inhibitors with possible applications for the therapy of solid tumors as well as autoimmune disorders.

In our screening efforts to identify unique scaffolds from myxobacteria for the drug discovery process, we used LC-SPE-NMR-MS techniques to isolate six linear peptides, termed macyranone A-F, from Cystobacter fuscus MCy9118. The macyranones are characterized by a rare 2-methylmalonamide moiety and an α-amino ketone fragment including an α',β'-epoxyketone in macyranone A. Gene disruption experiments confirmed the biosynthetic gene cluster of the macyranones as PKS/NRPS hybrid. Detailed in silico and phylogenetic analysis unraveled that the biosynthesis involves two conspicuous amide bond formations accomplished by an amidotransferase and a unique condensation domain. The gene cluster provides further insights into the formation of the powerful epoxyketone residue involving an acyl-CoA dehydrogenase and an unconventional free-standing thioesterase. Macyranone A was found to inhibit the chymotrypsin-like activity of the yeast 20S proteasome with an IC50 of 5.9 nM and the human constitutive proteasome and immunoproteasome with IC50 values of 21 and 15 nM, respectively. The β5 subunit of the 20S proteasome was characterized as target by X-ray crystallography revealing an irreversible binding mode similar to the natural product epoxomicin. The presence of the methylmalonamide residue facilitates the stabilization of macyranone A with the active β5 subunit of the proteasome. Macyranone A exhibits a potent inhibitory effect against the parasites Trypanosoma brucei rhodesiense and Leishmania donovani with IC50 values of 1.55 and 0.22 μM, respectively.