Nature of Pharmacophore Influences Active Site Specificity of Proteasome Inhibitors

Abstract

Proteasomes degrade most proteins in mammalian cells and are established targets of anti-cancer drugs. The majority of proteasome inhibitors are composed of short peptides with an electrophilic functionality (pharmacophore) at the C terminus. All eukaryotic proteasomes have three types of active sites as follows: chymotrypsin-like, trypsin-like, and caspase-like. It is widely believed that active site specificity of inhibitors is determined primarily by the peptide sequence and not the pharmacophore. Here, we report that active site specificity of inhibitors can also be tuned by the chemical nature of the pharmacophore. Specifically, replacement of the epoxyketone by vinyl sulfone moieties further improves the selectivity of ␤5-specific inhibitors NC-005, YU-101, and PR-171 (carfilzomib). This increase in specificity is likely the basis of the decreased cytotoxicity of vinyl sulfone-based inhibitors to HeLa cells as compared with that of epoxyketone-based inhibitors.The ubiquitin-proteasome pathway is essential in the maintenance of protein homeostasis in all eukaryotic cells and is involved in the regulation of numerous biologic processes. Proteasome inhibition causes apoptosis of malignant cells (1, 2). The proteasome inhibitor bortezomib (Velcade, PS-341) is used for the treatment of multiple myeloma and mantle cell lymphoma. Four other proteasome inhibitors are at different stages of clinical trials (3-6).The 26 S proteasome is a large (1.6 -2.4 MDa), hollow cylindrical, and multifunctional particle that consists of a 20 S proteolytic core and one or two 19 S regulatory complexes. Each eukaryotic 20 S core particle has three pairs of proteolytic sites with distinct substrate specificities (7-11). The ␤5 proteolytic sites are "chymotrypsin-like," and the ␤2 sites are "trypsin-like." The ␤1 sites cleave after acidic residues (Glu and Asp) and are referred to as "post-acidic," "post-glutamate peptide hydrolase," or "caspase-like." Tissues of the immune system also express immunoproteasomes, in which ␤5, ␤1, and ␤2 catalytic subunits are replaced by their major histocompatibility complex (MHC) locus-encoded counterparts LMP7 (␤5i), LMP2 (␤1i), and MECL-1 (␤2i).The chymotrypsin-like sites have long been considered the only suitable targets for anti-neoplastic agents and are the primary targets of all these agents. However, our recent work indicates that cytotoxicity of proteasome inhibitors correlates poorly with exclusive inhibition of the chymotrypsin-like sites and that co-inhibition of other sites is usually needed to achieve maximal cytotoxicity (12). In this regard, we have considered it of interest to determine whether inhibitors with increased specificity for ␤5 display decreased cytotoxicity.Many structural classes of proteasome inhibitors are known (2, 13). The majority of these are N-terminally capped short peptides (2-4 residues) with an electrophilic trap at the C terminus (e.g. aldehydes, boronates, epoxyketones, and vinyl sulfones). This electrophile reacts with the catalytic N-terminal threonines ...

One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor

“…This finding is also in agreement with their superior binding strength over MG132, which features an IC 50 value of 270 nM against the ChTL activity in vitro. Although it has been shown that inhibition of β5 alone is sufficient for cytotoxicity of a compound, it is considerably increased if also a second subunit is affected (27). This furthermore explains the enhanced effects of the syrbactin compounds, which bind both to the β5 and the β2 subunits.…”

Selected cytotoxic gold compounds cause significant inhibition of 20S proteasome catalytic activities

“…Targeting the CT-L active site has long been considered as sufficient to develop new candidate drugs for cancer treatment; yet, inhibition of multiple active sites is usually required to decrease markedly protein degradation and produce more relevant biological effects [5]. So, beyond the β5 active site, it is also important to target either the β1 or β2 active sites, considered as co-targets of cancer drugs [6,7,8]. Specific assays were developed to monitor independently the three individual catalytic activities of the proteasome and a number of selective inhibitors were identified [9,10,11].…”

Activity‐based probes for the multicatalytic proteasome

“…6) have a preference for hydrophobic and aromatic residues in most positions, consistent with the chymotrypsin-like activity of this subunit. Both VS and EK warheads have been evaluated, with EKs typically showing greater reactivity but reduced selectivity over b1 and b2 subunits [39,44]. Modification in the P1 and P3 positions can confer selectivity for either the b5c or b5i subunit: b5c favors a small hydrophobic group in P1 and a large hydrophobic group in P3, while b5i favors the reverse arrangement [35,40,45].…”