Peptide aldehydes are widely used as proteasome inhibitors in studies with cells in vitro
. N-terminal Z-capped tripeptide aldehydes typically inhibit the Mtb proteasome with IC50
values ranging from 10 – 70 µM [6
]. In unpublished work, we tested 20 N-Ac-Leu-Leu-Xaa-aldehydes against the Mtb proteasome (both wild type and open-gate mutant), where Xaa varied from small hydrophobic amino acids to aromatic amino acids. The IC50
values were similar and ranged from 25.8 µM to over 100 µM. They are classical Michaelis-Menton inhibitors. Surprisingly, the peptidomimetic 1
displayed time-dependent inhibition against both Mtb and human proteasomes. Compared to other peptide aldehydes, such as Z-Leu-Leu-Leu-Cho (ki
= 4 nM) [29
did not inhibit the mammalian proteasome any more potently, indicating that the elongated N-terminal capping group did not improve the overall binding affinity, but instead changed its kinetic behavior against the mammalian proteasome as seen in its slow onset of inhibition. However, 1
is over 1,000 fold more potent against the Mtb proteasome than the other peptide aldehydes that we have tested, indicating that elongation at the N-terminus dramatically changes the behavior of peptidyl aldehyde inhibitors, and may compensate for the unfavorable residue at the P1 position as well.
It is also surprising that 1
inhibits Mtb20S and hu20S with different kinetic mechanisms. Inhibition of Mtb20S by 1
follows a simple reversible slow-binding mechanism, whereas inhibition of hu20S β5 by 1
follows a two-step slow-binding mechanism. The one-step inhibition mechanism of 1
on the Mtb proteasome is supported by our crystal structure of the complex, which revealed no significant changes in the proteasome. The two-step mechanism of 1
on β5 of hu20S is consistent with the previously published structure of 1
-bound yeast proteasome, which revealed conformational changes in the core particle () [26
]. A Change in yeast proteasome structure upon inhibitor binding is interesting, because in our earlier report on oxathiazolones, the non-peptidic Mtb proteasome specific inhibitors, we observed marked conformational changes in Mtb proteasome following binding of oxathiazolones in the active site, whereas oxathiazolones did not inhibit the human proteasome [8
]. These inhibitor-induced structural changes hint at the potential plasticity of proteasomes. Indeed, structural flexibility might be required for proteasomes to achieve affinity for their diverse substrates in a cell. The conformational changes seen to date in eukaryotic and Mtb proteasomes are triggered by different small molecular scaffolds. More work is needed to better understand the differential structural responses to small molecule inhibitors between the seemingly well-conserved structures of Mtb and eukaryotic proteasomes.
In summary, we have identified fellutamide B as so far the most potent Mtb proteasome inhibitor. Fellutamide B inhibits Mtb 20S in a single step mechanism, while it inhibits the human proteasome in a two-step mechanism. The partial flexibility of the hydrophobic tail of 1 when complexed with the Mtb proteasome suggests that a shorter tail might further improve its binding for Mtb proteasome. Alternatively, the possible two binding pockets in the Mtb proteasome for the alky tail might allow for construction of more species-selective inhibitors with a branched tail that occupies both pockets simultaneously. A branched tail, while potentially advantageous for inhibition of the Mtb proteasome, might interfere with binding to human proteasomes. Studies in this direction are ongoing.