Structure-Based Design of Inhibitors Selective for Human Proteasome β2c or β2i Subunits

Abstract

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the β1c, β1i, β5c, and β5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting β2c or β2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the β2c- and β2i-selective compounds LU-002c (4; IC50 β2c: 8 nM, IC50 β2i/β2c: 40-fold) and LU-002i (5; IC50 β2i: 220 nM, IC50 β2c/β2i: 45-fold), respectively. Co-crystal structures with β2 humanized yeast proteasomes visualize protein–ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of β2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.

Two‐Step Bioorthogonal Activity‐Based Protein Profiling of Individual Human Proteasome Catalytic Sites

“…The synthesis of norbornene‐ABPs 11 , 13 , and 14 are also based on click ligation of norbornene‐alkyne 15 to their corresponding azide precursors (Figure ). The synthesis of 16 has been reported and the preparation of compounds 17 and 18 are described in the Supporting Information.…”

“…In the first instance, we assessed their subunit‐selectivity and activity by competitive ABPP making use of the activity‐based proteasome profiling assay and the structures of these probes are shown in Figure B . The activity, selectivity and cell permeability of azides 1 , 2 , 3 , and 5 in such assays had already been described. The results on the behavior of norbornenes 8 – 14 in competitive ABPP, in particular their activity, selectivity, and cell permeability are summarized in Figure and Tables and .…”

“…In total, the research described herein has expanded the proteasome ABP toolset to include five new in vitro ABPs, of which four can also be applied to monitor proteasome activity profiles in living cells. Further studies are required to complete a toolset for labeling each immunoproteasome and constitutive proteasome activity individually, this to complement our three‐probe multiplexing ABPP that is currently in use by us and others.…”

“…In case the inhibitors are covalent and irreversible, they can be modified to contain reporter entities for activity‐based proteasome profiling purposes. Recently, we disclosed a set of inhibitors selective for one of each of the six catalytic subunits of human constitutive proteasomes and immunoproteasomes (Figure ),– whereas activity‐based proteasome probes have also been reported by other research groups –. With this set of subunit‐selective inhibitors 1 – 7 (Figure A), each human proteasome activity can be inhibited at will.…”

Preparative Method for Asymmetric Synthesis of (S)-2-Amino-4,4,4-trifluorobutanoic Acid

“…As presented in Figure 1, the differences projected by several biphenyl rotational values are relatively modest, suggesting steric mimicry between these two groups, though the effective functional mimicry is very much dependent upon the actual biochemical settings [67]. In this context, the bioisosteric relationships between CF3 and i-Pr can be used for 2-amino-4,4,4-trifluorobutanoic acid 1 [68][69][70][71][72][73][74][75][76] substitution for leucine 2 in the de novo design of biologically active peptides and peptidomimetics [77][78][79][80][81][82][83]. As part of our ongoing research program focused on the application of fluorinated tailor-made AAs in pharmaceutical discovery, we needed a convenient access to large quantities of Fmoc-2amino-4,4,4-trifluorobutanoic acid 1 in both enantiomeric forms.…”