Incorporation of Non-natural Amino Acids Improves Cell Permeability and Potency of Specific Inhibitors of Proteasome Trypsin-like Sites

Abstract

Proteasomes degrade the majority of proteins in mammalian cells by a concerted action of three distinct pairs of active sites. The chymotrypsin-like sites are targets of antimyeloma agents bortezomib and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple myeloma cells to these agents. Here we describe systematic effort to develop inhibitors with improved potency and cell permeability, yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone (4a, LU-102), and a fluorescent activity-based probe for this site. X-ray structures of 4a and related inhibitors complexed with yeast proteasomes revealed the structural basis for specificity. Nontoxic to myeloma cells when used as a single agent, 4a sensitized them to bortezomib and carfilzomib. This sensitizing effect was much stronger than the synergistic effects of histone acetylase inhibitors or additive effects of doxorubicin and dexamethasone, raising the possibility that combinations of inhibitors of the trypsin-like site with bortezomib or carfilzomib would have stronger antineoplastic activity than combinations currently used clinically.

The novel 2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells

“…20 We hypothesized that additional inhibition of β2 proteasome activity in bortezomib-resistant myeloma cells would increase the degree of proteasome inhibition achieved by bortezomib alone, and re-sensitize them for bortezomib treatment via IRE-1a activation and induction of a terminal UPR. With this aim in view, we have developed the first synthetic proteasome inhibitor to inhibit the β2 activity, 21 and have improved its cell permeability and potency, yielding the compound LU-102, 22 which sensitizes RPMI8226 cells to bortezomib and carfilzomib-induced cytotoxicity. We now address the potential of LU-102 to overcome bortezomib/carfilzomib-resistance.…”

“…AMO-abtz/acfz cells were adapted to proteasome inhibitor-containing culture conditions as described. 20 The proteasome inhibitors bortezomib, carfilzomib and LU-102 22 were synthesized at the Leiden Institute of Chemistry. The selective inhibitors NC-001 (inhibiting β1/β1i) and NC-005 (β5/β5i) have been described by Britton et al…”

Activity‐based probes for the multicatalytic proteasome

“…The RLR recognition element was more active against purified proteasomes, but less active in cells, presumably due to poor cell permeability. Geurink et al subsequently reported that vinyl sulfones are more potent and selective toward the b2 subunits than their epoxyketone analogs [43]. Furthermore, replacement of arginine with the non-natural amino acid 4-(aminomethyl) phenylalanine in either the P1 site (LU012) [43] or P1 and P3 sites (LU112) further improved affinity and chemical stability.…”

“…These compounds have an azide handle that can be used for attachment of biotin or a fluorophore after labeling. However, introduction of the fluorophore prior to labeling reduced selectivity over the b5 subunit [43]. Despite the trypsin-like selectivity of b5, a basic residue in the P1 site is not required.…”

“…For example, azide-modified analogs of the subunit-specific inhibitors NC-001, NC-002, and NC-005 were used to demonstrate that the compounds were highly specific for the proteasome [38,42]. The small azide label had minimal effect on activity, unlike larger fluorophore labels that can adversely affect activity or selectivity [43]. However, labeling was inferred from an anti-biotin blot, which lacks sensitivity.…”