Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

BACKGROUND: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits. RESULTS: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(methyl)-sulfone (AdaAhx(3)L(3)VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. CONCLUSIONS: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts.     

A Set of Activity‐Based Probes to Visualize Human (Immuno)proteasome Activities

Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, β1c, β2c, and β5c. Lymphoid tissues also express the immunoproteasome subunits β1i, β2i, and β5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity‐based probes that enables simultaneous gel‐based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for β1c, β2c, β5c, and β2i, to compare the active‐site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of β5i and β1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients.     

Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells

Background: Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities.Results: Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aidehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called ‘branched chain amino acid preferring’ (BrAAP) and ‘small neutral amino acid preferring’ (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity.Conclusions: The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate,     

Natural compounds with proteasome inhibitory activity for cancer prevention and treatment

The proteasome is a multicatalytic protease complex that degrades most endogenous proteins including misfolded or damaged proteins to ensure normal cellular function. The ubiquitin-proteasome degradation pathway plays an essential role in multiple cellular processes, including cell cycle progression, proliferation, apoptosis and angiogenesis. It has been shown that human cancer cells are more sensitive to proteasome inhibition than normal cells, indicating that a proteasome inhibitor could be used as a novel anticancer drug. Indeed, this idea has been supported by the encouraging results of the clinical trials using the proteasome inhibitor Bortezomib (Velcade, PS-341), a drug approved by the US Food and Drug Administration (FDA). Several natural compounds, including the microbial metabolite lactacystin, green tea polyphenols, and traditional medicinal triterpenes, have been shown to be potent proteasome inhibitors. These findings suggest the potential use of natural proteasome inhibitors as not only chemopreventive and chemotherapeutic agents, but also tumor sensitizers to conventional radiotherapy and chemotherapy. In this review, we will summarize the structures and biological activities of the proteasome and several natural compounds with proteasome inhibitory activity, and will discuss the potential use of these compounds for the prevention and treatment of human cancers.     

Molecular modeling for the interaction between proteasome beta 5 subunit and organotin compounds

It has been reported that organotins can inhibit the proteasomal chymotrypsin-like activity and induce cell death, but the interaction mode of organotins with proteasome has not been well defined. In this study, the IC₅₀ of butyltins and phenyltins against the proteasomal activity and the nature of their inhibition were investigated. It was found that both mono- and di-organotins were weak, reversible inhibitors against the proteasome, while tributyltin and triphenyltin were potent, irreversible proteasome inhibitors. In silico studies using the reversible organotin proteasome inhibitors demonstrated a tight correlation of the estimated proteasomal inhibition constants (Ki) with the experimental IC₅₀ values for proteasome inhibition. Furthermore, the Sn atom in TBT and TPT was found susceptible to form a coordinate bond with Thr 1 O^γ of the β5 subunit, which may account for the irreversible proteasome inhibition. The computational docking approach well predicted the inhibition nature of organotins toward the proteasomal chymotrypsin-like activity. This predictive model might aid in understanding the cytotoxic behavior of similar organometallic compounds.     

Native FKBP12 engineering by ligand-directed tosyl chemistry: labeling properties and application to photo-cross-linking of protein complexes in vitro and in living cells

The ability to modify target "native" (endogenous) proteins selectively in living cells with synthetic molecules should provide powerful tools for chemical biology. To this end, we recently developed a novel protein labeling technique termed ligand-directed tosyl (LDT) chemistry. This method uses labeling reagents in which a protein ligand and a synthetic probe are connected by a tosylate ester group. We previously demonstrated its applicability to the selective chemical labeling of several native proteins in living cells and mice. However, many fundamental features of this chemistry remain to be studied. In this work, we investigated the relationship between the LDT reagent structure and labeling properties by using native FK506-binding protein 12 (FKBP12) as a target protein. In vitro experiments revealed that the length and rigidity of the spacer structure linking the protein ligand and the tosylate group have significant effects on the overall labeling yield and labeling site. In addition to histidine, which we reported previously, tyrosine and glutamate residues were identified as amino acids that are modified by LDT-mediated labeling. Through the screening of various spacer structures, piperazine was found to be optimal for FKBP12 labeling in terms of labeling efficiency and site specificity. Using a piperazine-based LDT reagent containing a photoreactive probe, we successfully demonstrated the labeling and UV-induced covalent cross-linking of FKBP12 and its interacting proteins in vitro and in living cells. This study not only furthers our understanding of the basic reaction properties of LDT chemistry but also extends the applicability of this method to the investigation of biological processes in mammalian cells.     

Specific cell-permeable inhibitor of proteasome trypsin-like sites selectively sensitizes myeloma cells to bortezomib and carfilzomib

Proteasomes degrade the majority of proteins in mammalian cells, are involved in the regulation of multiple physiological functions, and are established targets of anticancer drugs. The proteasome has three types of active sites. Chymotrypsin-like sites are the most important for protein breakdown and have long been considered the only suitable targets for antineoplastic drugs; however, our recent work demonstrated that inhibitors of caspase-like sites sensitize malignant cells to inhibitors of the chymotrypsin-like sites. Here, we describe the development of specific cell-permeable inhibitors and an activity-based probe of the trypsin-like sites. These compounds selectively sensitize multiple myeloma cells to inhibitors of the chymotrypsin-like sites, including antimyeloma agents bortezomib and carfilzomib. Thus, trypsin-like sites are cotargets for anticancers drugs. Together with inhibitors of chymotrypsin- and caspase-like sites developed earlier, we provide the scientific community with a complete set of tools to separately modulate proteasome active sites in living cells.     

Patented small molecule inhibitors in the ubiquitin proteasome system

Deregulation of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. The recent approval of the proteasome inhibitor Velcade(R) (bortezomib) for the treatment of multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. We review here new patented proteasome inhibitors and patented small molecule inhibitors targeting more specific UPS components, such as E3 ubiquitin ligases and deubiquitylating enzymes. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Towards subunit-specific proteasome inhibitors: synthesis and evaluation of peptide alpha',beta'-epoxyketones

BACKGROUND: The proteasome is a large multicatalytic protease complex (700 kDa) involved in a number of highly regulated processes. It has three major catalytic activities: a chymotrypsin-like activity, a trypsin-like activity and a post-glutamyl peptide hydrolyzing (PGPH) activity. To be useful as molecular probes, which could help dissect the cellular functions of the proteasome, inhibitors should be specific for the proteasome, active in vivo and selectively block only one of the three catalytic activities. To date, few inhibitors fulfill these requirements so we set out to make novel proteasome inhibitors that incorporate these characteristics. RESULTS: A panel of amino-terminally acetylated peptide alpha',beta'-epoxyketones with leucine in P1 and various aliphatic or aromatic amino acids in P2-P4 were prepared and evaluated. Most compounds selectively inhibited the chymotrypsin-like activity, while only weakly inhibiting the trypsin-like and PGPH activities. After optimization, one inhibitor, Ac-hFLFL-epoxide, was found to be more potent and selective for the inhibition of the chymotrypsin-like activity than several previously described inhibitors. This inhibitor also exhibited strong in vivo anti-inflammatory activity. CONCLUSIONS: Optimization of amino-terminally acetylated peptide alpha',beta'-epoxyketones furnished a potent proteasome inhibitor, Ac-hFLFL-epoxide, that has an excellent selectivity for the chymotrypsin-like activity. The inhibitor also proved to be a potent antiproliferative and anti-inflammatory agent. The strong in vivo and in vitro activities suggest that this class of proteasome inhibitors could be both molecular probes and therapeutic agents.     

Differential protein expression of hepatic cells associated with MeHg exposure: deepening into the molecular mechanisms of toxicity

Understanding the molecular mechanisms underlying MeHg toxicity and the way in which this molecule interacts with living organisms is a critical point since MeHg represents a well-known risk to ecosystems and human health. We used a quantitative proteomic approach based on stable isotopic labeling by amino acids in cell culture in combination with SDS-PAGE and nanoflow LC-ESI-LTQ for analyzing the differential protein expression of hepatic cells associated to MeHg exposure. Seventy-eight proteins were found de-regulated by more than 1.5-fold. We identified a number of proteins involved in different essential biological processes including apoptosis, mitochondrial dysfunction, cellular trafficking and energy production. Among these proteins, we found several molecules whose de-regulation has been already related to MeHg exposure, thus confirming the usefulness of our discovery approach, and new ones that helped to gain a deeper insight into the biomolecular mechanisms related to MeHg-induced toxicity. Overexpression of several HSPs and the proteasome 26S subunit itself showed the proteasome system as a molecular target of toxic MeHg. As for the interaction networks, the top ranked was the nucleic acid metabolism, where many of the identified de-regulated proteins are involved.     

Identification of the Binding Site of Brucella VirB8 Interaction Inhibitors

Secretion systems translocate virulence factors of many bacterial pathogens, enabling their survival inside the host organism. Consequently, inhibition strongly attenuates pathogenicity and can be considered a target for novel antimicrobial drugs. The type IV secretion system (T4SS) of the intracellular pathogen Brucella is a prerequisite for its virulence, and in this work we targeted the interactions of the?essential assembly factor protein, VirB8, using small-molecule inhibitors. High-throughput screening identified several potent and specific inhibitors, and the target-binding site of these inhibitors was identified by X-ray crystallography, in?silico docking, and analysis of the derivates of the inhibitor B8I-2. VirB8 interaction inhibitors bind to a surface groove opposite to the dimerization interface, and by varying the binding-site residues, we were able to determine which residues are required for inhibitor activity. E115 and K182 were found to be especially important, and changes at R114, Y229, and L151 also reduced inhibitor efficiency.     

Tunable Probes with Direct Fluorescence Signals for the Constitutive and Immunoproteasome

Electrophiles are commonly used for the inhibition of proteases. Notably, inhibitors of the proteasome, a central determinant of cellular survival and a target of several FDA‐approved drugs, are mainly characterized by the reactivity of their electrophilic head groups. We aimed to tune the inhibitory strength of peptidic sulfonate esters by varying the leaving groups. Indeed, proteasome inhibition correlated well with the pK_a of the leaving group. The use of fluorophores as leaving groups enabled us to design probes that release a stoichiometric fluorescence signal upon reaction, thereby directly linking proteasome inactivation to the readout. This principle could be applicable to other sulfonyl fluoride based inhibitors and allows the design of sensitive probes for enzymatic studies.     

Inhibitors for the immuno- and constitutive proteasome: current and future trends in drug development

Proteolytic degradation is an essential cellular process which is primarily carried out by the 20S proteasome core particle (CP), a protease of 720 kDa and 28 individual subunits. As a result of its central functional role, the proteasome represents an attractive drug target that has been extensively investigated during the last decade and validated by the approval of bortezomib by the US Food and Drug Administration (FDA). Currently, several optimized second‐generation proteasome inhibitors are being explored as anticancer drugs in clinical trials, and most of them target both constitutive proteasomes (cCPs) and immunoproteasomes (iCPs). However, selective inhibition of the iCPs, a distinct class of proteasomes predominantly expressed in immune cells, appears to be a promising therapeutic rationale for the treatment of autoimmune disorders. Although a few selective agents have already been identified, the recently determined crystal structure of the iCP will further promote the development and optimization of iCP‐selective compounds.     

Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes

Background: The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active β subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.Results: A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 1251-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of β-subunit modification.Conclusions: The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual β subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual β subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.     

Validation of the Proteasome as a Therapeutic Target in Plasmodium Using an Epoxyketone Inhibitor with Parasite-Specific Toxicity

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Highlights? Purification of Plasmodium proteasomes and labeling with an activity-based probe ? Identification of an epoxyketone-based inhibitor with parasite-specific toxicity ? Demonstration that Plasmodium is sensitive to partial inhibition of its proteasome ? Determination of key parameters for selective parasite killing with proteasome inhibitors     

Discovery of a potent and highly β1 specific proteasome inhibitor from a focused library of urea-containing peptide vinyl sulfones and peptide epoxyketones

Syringolins, a class of natural products, potently and selectively inhibit the proteasome and show promising antitumour activity. To gain insight in the mode of action of syringolins, the ureido structural element present in syringolins is incorporated in oligopeptide vinyl sulfones and peptide epoxyketones yielding a focused library of potent new proteasome inhibitors. The distance of the ureido linkage with respect to the electrophilic trap strongly influences subunit selectivity within the proteasome. Compounds 13 and 15 are β5 selective and their potency exceeds that of syringolin A. In contrast, 5 may well be the most potent β1 selective compound active in living cells reported to date.     

O-GlcNAcylation mapping of single living cells by in situ quantitative SERS imaging

O-GlcNAcylation is involved in many biological processes including cancerization. Nevertheless, its in situ quantification in single living cells is still a bottleneck. Here we develop a quantitative SERS imaging strategy for mapping the O-GlcNAcylation distribution of single living cells. O-GlcNAcylated compounds (OGCs) can be quantified through their in situ azide labeling and then a click reaction competing with azide and Raman reporter labeled 15 nm-gold nanoparticles (AuNPs) for linking to dibenzocyclooctyne labeled 40 nm-AuNPs to produce OGC-negatively correlated SERS signals. The calibration curve obtained in vitro can be conveniently used for detecting OGCs in different areas of single living cells due to the negligible effect of cell medium on the click linkage and Raman signal. This method has been successfully applied in mapping O-GlcNAcylation distribution in different cell lines and monitoring O-GlcNAcylation variation during cell cycling, which demonstrate its great practicability and expansibility in glycosylation related analysis.

A quantitative SERS imaging strategy is developed for O-GlcNAcylation mapping of single living cells through a competitive click reaction.     

Joining the army of proteasome inhibitors

An article in this issue of Chemistry & Biology (Hines et al., 2008) and a recent study in Nature (Groll et al., 2008) establish three natural products as novel proteasome inhibitors. These inhibitors, discovered in an unusual way, reveal a different mechanism of proteasome inhibition and suggest new therapeutic application of its inhibitors.     

Determination of intracellular prolyl/glycyl proteases in intact living human cells and protoporphyrin IX production as a reporter system

The determination of enzyme activity or inhibition in intact living cells is a problem in the development of inhibitors for intracellular proteases. The production of fluorescent protoporphyrin IX (PpIX) from the nonfluorescent (N)-Gly/Pro-5-aminolevulinic acid (ALA) substrates was used to evaluate the prolyl/glycyl-specific dipeptidylpeptidase IV (DPPIV)-like and prolyloligopeptidase (POP)-like activities of human cells. The results demonstrated that whereas POP-like activity could be attributed to the actual POP, the DPPIV-like activity could be related to actual DPPIV only in one colon cell line. In the other breast and colon cell lines, DPPIV-like activity was intracellular and displayed by other prolyl-specific aminopeptidases. Our experiments also demonstrated the involvement of glycyl-specific proteases in the processing of ALA precursors. These observations have important consequences for the development and evaluation of selective inhibitors for these enzymes.