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Abstract

Inhibition of the immunoproteasome subunit β5i alleviates autoimmune diseases in preclinical studies and represents a promising new anti-inflammatory therapy. However, the lack of structural data on the human immunoproteasome still hampers drug design. Here, we systematically determined the potency of seven α' β' epoxyketone inhibitors with varying N-caps and P3-stereochemistry for mouse/human β5c/β5i and found pronounced differences in their subunit and species selectivity. Using X-ray crystallography, the compounds were analyzed for their modes of binding to chimeric yeast proteasomes that incorporate key parts of human β5c, human β5i or mouse β5i and the neighboring β6 subunit. The structural data reveal exceptional conformations for the most selective human β5i inhibitors and highlight subtle structural differences as the major reason for the observed species selectivity. Altogether, the presented results validate the humanized yeast proteasome as a powerful tool for structure-based development of β5i inhibitors with potential clinical applications.

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... To resolve the binding mode of the ligands to the proteasomal active site and their basis for selectivity, the most potent (IC 50 value of 0.025 µM for human β5i) and selective (800-fold over β5i) compound of this series [81], termed Ro19 (Figure 3), was studied by X-ray crystallography with a humanized yeast proteasome, featuring the human β5i/β6-substrate binding channel [105]. The X-ray data visualized that Ro19 occupies the S1-pocket and also S1/S3-sub-sites that are not accessible to natural peptide-based ligands but various synthetic compounds [106,107]. Ro19 and all other compounds of this class are β5i-selective, as their bulky quinolone moiety complies better with the spacious S1-pocket of β5i than with the smaller one of β5c. In addition, binding to the active sites of β1i and β2i is precluded by steric hindrance of the thiazole ring with residues of their S3-pockets. ...
... Thus, inhibitors with oversized P1-sites or non-standard binding modes like Ro19 may be associated with significant species-selectivity and demand for careful selection of animal models for preclinical studies. Similar observations were made with the covalent β5i-inhibitors PR-924 [106] and M3258 [66] (see Section 4.3.2.) ...
... As initial structural data could not explain this difference in performance [73], X-ray crystallographic data with humanized yeast proteasomes were collected. The structures visualized distinct binding modes for PR-924 in β5c/β6and β5i/β6-substrate binding channels, respectively [106]. Consistent with the non-natural stereochemistry at P3, PR-924 adopts a kinked conformation in humanized β5i-proteasome structures, with the N-cap occupying a S3*-sub-pocket that cannot be approached by natural ligands. ...
Article
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At the heart of the ubiquitin–proteasome system, the 20S proteasome core particle (CP) breaks down the majority of intracellular proteins tagged for destruction. Thereby, the CP controls many cellular processes including cell cycle progression and cell signalling. Inhibitors of the CP can suppress these essential biological pathways, resulting in cytotoxicity, an effect that is beneficial for the treatment of certain blood cancer patients. During the last decade, several preclinical studies demonstrated that selective inhibition of the immunoproteasome (iCP), one of several CP variants in mammals, suppresses autoimmune diseases without inducing toxic side effects. These promising findings led to the identification of natural and synthetic iCP inhibitors with distinct chemical structures, varying potency and subunit selectivity. This review presents the most prominent iCP inhibitors with respect to possible scientific and medicinal applications, and discloses recent trends towards pan-immunoproteasome reactive inhibitors that cumulated in phase II clinical trials of the lead compound KZR-616 for chronic inflammations.
... To establish the apparent IC 50 values more accurately and to obtain insights into the coinhibition of β1c, β1i, β5c, and β5i activities, we selected the compounds 4, 7, 13, 16,18,20,22, and 25 for further analysis. In our competitive ABPP assay using Raji cell extracts (containing both cCPs and iCPs), a wider range of final concentrations were tested. ...
... As structural data on human apo iCP are not available, we recently developed chimeric yeast proteasomes, which feature the key elements of human β5 subunits, as structural tools. 18 On the basis of this work, we created here β2 humanized yeast proteasomes. ...
... Although the yeast proteasome (yCP) α subunits can be easily exchanged by human counterparts, the replacement of most β entities, that is, β1, β2, β5, β6, and β7 is lethal to yeast. 13d, 18,19 Strikingly, however, the single-point mutation S171G suffices to rescue the lethal phenotype that is caused by the substitution of the endogenous yeast (y) β2 subunit with the human (h) β2c counterpart. 19 We created the respective ...
Article
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Subunit-selective proteasome inhibitors are valuable tools to assess the biological function and medicinal relevance of individual proteasome active sites in a specific context. While inhibitors for the β1c, β1i, β5c and β5i subunits exploit structural differences in the substrate binding channels identified by X-ray crystallography, compounds selectively targeting β2c or β2i could not be rationally designed so far due to the high degree of structural similarity of these 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: 20-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, an elaborate combination of organic syntheses, activity-based protein profiling, yeast mutagenesis and structural biology allowed us to decipher subtle but significant differences of β2 substrate binding channels and to complete the set of subunit-selective proteasome inhibitors by identifying β2c- and β2i-selective compounds.
... Notably, the β5 S1 site methionine (Met76) is retracted, potentially due to van der Waals interactions with adjacent Tyr and His residues in β6 35 , creating a larger binding pocket (Supplementary Fig. 4). Previous work has established that the relative orientation of this methionine (Met45 in yeast) is important for the binding of several inhibitors 35,36 . In fact, several selective proteasome inhibitors like PR-957 have a 15-to 255-fold higher affinity for mammalian immunoproteasomes than for constitutive proteasomes 35,36 . ...
... Previous work has established that the relative orientation of this methionine (Met45 in yeast) is important for the binding of several inhibitors 35,36 . In fact, several selective proteasome inhibitors like PR-957 have a 15-to 255-fold higher affinity for mammalian immunoproteasomes than for constitutive proteasomes 35,36 . The methionine of immunoproteasomes is in an 'open' conformation, allowing the efficient insertion of a bulky sidechain into the S1 site. ...
Article
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Proteasomes play an essential role in the life cycle of intracellular pathogens with extracellular stages by ensuring proteostasis in environments with limited resources. In microsporidia, divergent parasites with extraordinarily streamlined genomes, the proteasome complexity and structure are unknown, which limits our understanding of how these unique pathogens adapt and compact essential eukaryotic complexes. We present cryo-electron microscopy structures of the microsporidian 20S and 26S proteasome isolated from dormant or germinated Vairimorpha necatrix spores. The discovery of PI31-like peptides, known to inhibit proteasome activity, bound simultaneously to all six active sites within the central cavity of the dormant spore proteasome, suggests reduced activity in the environmental stage. In contrast, the absence of the PI31-like peptides and the existence of 26S particles post-germination in the presence of ATP indicates that proteasomes are reactivated in nutrient-rich conditions. Structural and phylogenetic analyses reveal that microsporidian proteasomes have undergone extensive reductive evolution, lost at least two regulatory proteins, and compacted nearly every subunit. The highly derived structure of the microsporidian proteasome, and the minimized version of PI31 presented here, reinforce the feasibility of the development of specific inhibitors and provide insight into the unique evolution and biology of these medically and economically important pathogens.
... After visual inspection, availability of derivatives, and synthetic feasibility, we selected 2-vinylthiazole (XXI, RA (β5i) = 5 ± 3%, Figure 4) to be incorporated into a potential covalent inhibitor containing the additional boronic acid warhead moiety similar to the (R)-boroleucine moiety of bortezomib (XXII, Figure 4). The compound was designed based on the X-ray structure of bortezomib in complex with humanized yeast proteasome [39,40], with the vinylthiazole group added at a position that could allow its access to the β5i-specific Cys48 residue. Details of the synthesis can be found in the Supplementary Materials (Scheme S5). ...
... In an attempt to investigate the mechanism of inhibition, the Ellman's assay to determine the fraction of available cysteines was performed using β5i with and without incubation of the compound. However, we found that the Ellman's assay was not feasible with bidentate compounds (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) due to significant assay interference. In contrast, intact MS/MS experiments showed single labelling of β5i by 39 ( Figure S4). ...
Article
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Constitutive- and immunoproteasomes are part of the ubiquitin–proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the β5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and β5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the β5, β5i, β1, and β1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either β5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.
... The phenylethyl group at P1 perfectly fits into the spacious S1 pocket of b5i andi ss tabilized by ac ation-p contact with K33, a sulfur-arene interaction with M45, [32,33] and by CÀH-p interactions with the side chains of M31, K33, and A49. Interestingly, the overlay of the docked pose of 21 with the b5i-specific inhibitors PR-957( PDB ID:3 UNF) and PR-924b ound to the chimeric hb5-hb6s ubstrate binding channel (PDB ID:5 L5H) [34] reveals that 21 and PR-924 adopt as imilar kinked bindingm ode, with an identical positioning of the amide scaffolds and as imilar orientation of the P1 and P3 functions, whereas PR-957 adopts al inear orientation ( Figure 3D). The superior b5i selectivity of 21 over ligands that target the strictly conserved peptide binding sites seems to resultfrom its ability to exploit subpockets other than the substrate binding channels. ...
... Protein and ligand preparation:B ecause the only available crystal structures of the human i20S are yeast chimera that incorporate key parts of human b5i and the neighboring b6s ubunit but are devoid of the b1i subunit, [34] we used the crystal structure of the murine i20S in complex with the inhibitor PR-957 (PDB ID:3 UNF) [7] for docking studies. Murine and human 20S subunits share as equence identity of more than 90 %, and the few non-identical residues are external to the active sites. ...
Article
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The development of immunoproteasome‐selective inhibitors is a promising strategy for treating hematologic malignancies, autoimmune and inflammatory diseases. In this context, we report the design, synthesis, and biological evaluation of a new series of amide derivatives as immunoproteasome inhibitors. Notably, the designed compounds act as noncovalent inhibitors, which might be a promising therapeutic option because of the lack of drawbacks and side effects associated with irreversible inhibition. Among the synthesized compounds, we identified a panel of active inhibitors with Ki values in the low micromolar or sub‐micromolar ranges toward the β5i and/or β1i subunits of immunoproteasomes. One of the active compounds was shown to be the most potent and selective inhibitor with a Ki value of 21 nm against the single β1i subunit. Docking studies allowed us to determine the mode of binding of the molecules in the catalytic site of immunoproteasome subunits.
... 21 At the outset of our work, PR-924 was a known, selective inhibitor of human LMP7 (Table 1). 22 Our model and recent cocrystal structures in humanized yeast proteasome 23 suggested selectivity for LMP7 arose from interactions between PR-924's indene terminus and the LMP7 S3 pocket formed at neighboring β6. In contrast to ONX 0914 binding, a change in stereochemistry at P3 places PR-924's terminal carboxamide deep within the S3 pocket ( Figure 2b). ...
... (S)-2-(tert-Butoxycarbonylamino)-3-cyclopentenylpropanoic Acid (23). To a solution of cyclopentanone (55.0 g, 0.665 mol) in DCM (1.3 L) was added Na 2 CO 3 (104 g, 0.980 mol), and the mixture was cooled to −20°C. ...
Article
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Selective immunoproteasome inhibition is a promising approach for treating autoimmune disorders, but optimal proteolytic active site subunit inhibition profiles remain unknown. We reveal here our design of peptide epoxyketone-based selective LMP7 and MECL-1 subunit inhibitors. Utilizing these and our previously disclosed LMP2 inhibitor, we demonstrate a requirement of dual LMP7/LMP2 or LMP7/MECL-1 subunit inhibition profiles for potent cytokine expression inhibition and in vivo efficacy in an inflammatory disease model. These and additional findings toward optimized solubility led the design and selection of KZR-616 disclosed here and presently in clinical trials for treatment of rheumatic disease.
... These assays indicate that inhibitors selective for human LMP7 are not naturally selective for mouse proteasomes. Indeed, X-ray structures of humanized yeast proteasome identify unique binding modes of inhibitors for human β5i (Huber, Heinemeyer, de Bruin, Overkleeft & Groll, 2016). Although the substrate binding pockets and the active site residues are highly conserved among mouse and human, the residues outside the natural substrate binding channel, such as the identified S3* pocket, are subjected to variances, thereby creating species-specific differences such as Val/Met31 (Huber, Heinemeyer, de Bruin, Overkleeft & Groll, 2016). ...
... Indeed, X-ray structures of humanized yeast proteasome identify unique binding modes of inhibitors for human β5i (Huber, Heinemeyer, de Bruin, Overkleeft & Groll, 2016). Although the substrate binding pockets and the active site residues are highly conserved among mouse and human, the residues outside the natural substrate binding channel, such as the identified S3* pocket, are subjected to variances, thereby creating species-specific differences such as Val/Met31 (Huber, Heinemeyer, de Bruin, Overkleeft & Groll, 2016). Based on the activity in the mouse proteasome we decided to further investigate two compounds, LU-005i and LU-001i, in more detail. ...
Article
Background and purpose: MECL-1 (β2i), LMP2 (β1i), and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome, a special type of proteasome mainly expressed in hematopoietic cells. Targeting LMP7 has been shown to be therapeutically effective in pre-clinical models of autoimmune diseases. In this study, a recently described inhibitor of the immunoproteasome LU-005i was investigated with respect to selectivity and biological activity. Experimental approach: The specificity of LU-005i and other immunoproteasome selective inhibitors was characterized using fluorogenic peptide substrates. The effect of proteasome inhibition on cytokine release was investigated with endotoxin-stimulated mouse splenocytes or human PBMCs. Measurement of weight loss and colon length was used to analyse the effect of proteasome inhibition on inflammatory bowel disease in the dextran sulfate sodium (DSS)-induced colitis model. Key results: LU-005i is the first human and mouse immunoproteasome selective inhibitor targeting all three proteolytically active immunoproteasome subunits. Cytokine secretion of LU-005i-exposed endotoxin-stimulated mouse splenocytes or human PBMCs was strongly reduced. Furthermore, differentiation of naïve T helper cells to T helper 17 cells was impaired in the presence of LU-005i. Additionally, LU-005i ameliorated DSS-induced colitis. Conclusion and implications: This study with a novel pan-immunoproteasome inhibitor substantiates that the immunoproteasome is a promising drug target for the treatment of inflammatory diseases and that exclusive inhibition of LMP7 is not necessary for therapeutic effectiveness. Our results will promote the design of new generations of immunoproteasome inhibitors with optimal therapeutic efficacy for clinical use in the treatment of autoimmunity and cancer.
... Previous work showed that M3258 displayed significantly reduced in vitro inhibition of LMP7 in mouse cells, compared with its activity in human, rat, dog, and monkey cells . As described previously (Huber et al., 2016;Sanderson et al., 2021) the comparable inhibitory activity of M3258 against human, rat, dog, and monkey LMP7 is likely explained by the highly conserved active site sequence of these orthologs. Meanwhile the presence of a bulky methionine-31 in the mouse LMP7 active site (valine in human, rat, dog, and monkey LMP7) results in reduced activity of selective LMP7 inhibitors in mouse cells. ...
Article
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M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, β5i, PSMB8) proteolytic subunit of the immunoproteasome; a component of the cellular protein degradation machinery, highly expressed in malignant hematopoietic cells including multiple myeloma. Here we describe the fit-for-purpose PK/PD/efficacy modelling of M3258 based on preclinical data from several species. The inhibition of LMP7 activity (PD) and tumor growth (efficacy) were tested in human multiple myeloma xenografts in mice. PK and efficacy data were correlated yielding a free M3258 concentration of 45 nM for half-maximal tumor growth inhibition (KC50). Because inhibition of LMP7 could only hardly be detected in mouse PBMCs, both in vitro and in vivo bridging studies were performed in rats, monkeys, and dogs for translational modelling. These data indicated that the PD response in human xenograft models was closely reflected in dog PBMCs. A PK/PD model was established, predicting a free IC50 value of 9 nM for M3258 in dogs in vivo, in close agreement to in vitro measurements. In parallel, the human PK parameters of M3258 were predicted by various approaches including in vitro extrapolation and allometric scaling. Using PK/PD/efficacy simulations, the efficacious dose range and corresponding PD response in human were predicted. Taken together, these efforts supported the design of a phase Ia study of M3258 in multiple myeloma patients (NCT04075721). At the lowest tested dose level, the predicted exposure matched well with the observed exposure while the duration of LMP7 inhibition was underpredicted by the model. Significance Statement M3258 is a novel inhibitor of the immunoproteasome subunit LMP7. The human PK and human efficacious dose range of M3258 were predicted using in vitro - in vivo extrapolation and allometric scaling methods together with a fit-for-purpose PK/PD and efficacy model based on data from several species. A comparison with data from the Phase Ia clinical study showed that the human PK was accurately predicted, whilst the extent and duration of PD response were more pronounced than estimated.
... Replacement of L-Ala in the P3 position with D-Ala and of N-terminal morpholino group with a 3-methyl-H-indene reduced inhibition of β5c and β1i sites, leading to a highly selective β5i inhibitor PR-924 (IPSI) [61]. However, PR-924 is a less potent inhibitor of β5i in a mouse because of subtle differences in substrate binding pockets between murine and human immunoproteasomes [43,62]. Replacement of the phenyl moiety in the P1 position of PR-924 with a cyclohexyl moiety increased β5i selectivity even further, generating LU-015i [63]. ...
Article
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Proteasome is a multi-subunit protein degradation machine, which plays a key role in the maintenance of protein homeostasis and, through degradation of regulatory proteins, in the regulation of numerous cell functions. Proteasome inhibitors are essential tools for biomedical research. Three proteasome inhibitors, bortezomib, carfilzomib, and ixazomib are approved by the FDA for the treatment of multiple myeloma; another inhibitor, marizomib, is undergoing clinical trials. The proteolytic core of the proteasome has three pairs of active sites, β5, β2, and β1. All clinical inhibitors and inhibitors that are widely used as research tools (e.g., epoxomicin, MG-132) inhibit multiple active sites and have been extensively reviewed in the past. In the past decade, highly specific inhibitors of individual active sites and the distinct active sites of the lymphoid tissue-specific immunoproteasome have been developed. Here, we provide a comprehensive review of these site-specific inhibitors of mammalian proteasomes and describe their utilization in the studies of the biology of the active sites and their roles as drug targets for the treatment of different diseases.
... In contrast, bortezomib displayed comparable potency of LMP7 inhibition in this mouse cell line (IC 50 ¼ 5.3 nmol/L) and cells from other species. This weaker activity of M3258 toward mouse LMP7 could be explained by the presence of a bulky methionine-31 in mouse LMP7 (substituted by valine in human LMP7, as indicated in Fig. 1C, and also in rat and dog LMP7), which was previously described to sterically hinder interaction with partially selective LMP7 inhibitors, while still accommodating bortezomib binding (26). ...
Article
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Large multifunctional peptidase 7 (LMP7/β5i/PSMB8) is a proteolytic subunit of the immunoproteasome, which is predominantly expressed in normal and malignant hematolymphoid cells, including multiple myeloma, and contributes to the degradation of ubiquitinated proteins. Described herein for the first time is the preclinical profile of M3258; an orally bioavailable, potent, reversible and highly selective LMP7 inhibitor. M3258 demonstrated strong antitumor efficacy in multiple myeloma xenograft models, including a novel model of the human bone niche of multiple myeloma. M3258 treatment led to a significant and prolonged suppression of tumor LMP7 activity and ubiquitinated protein turnover and the induction of apoptosis in multiple myeloma cells both in vitro and in vivo. Furthermore, M3258 showed superior antitumor efficacy in selected multiple myeloma and mantle cell lymphoma xenograft models compared with the approved nonselective proteasome inhibitors bortezomib and ixazomib. The differentiated preclinical profile of M3258 supported the initiation of a phase I study in patients with multiple myeloma (NCT04075721).
... The affinity of the tested compounds toward β5i was observed in making favorable interactions with critical residues, i.e. Thr1, Gly47, Tyr169 and Ser130, which have roles in inflammatory responses [26]. The details of the PLI profile are listed in Table 2. ...
Article
Inflammatory bowel disease is a chronic and pathologic autoimmune condition. And immunoproteasome is becoming an attractive therapeutic target for autoimmune inflammatory diseases. In this study, we evaluated the therapeutic effects of a specific small molecule inhibitor of the chymotryptic-like β5i subunits of the immunoproteasome, DPLG3, in a preclinical murine colitis model and explored the underlying molecular mechanism for the immune suppression. DPLG3 showed significant effects in attenuating the disease progression in experimental colitis, reducing the body and spleen weight losses, and colon length shortening compared to vehicle-treated controls and to the well studied immunoproteasome inhibitor ONX-0914. Mechanistically, DPLG3 decreased inflammatory cytokines and the influx of effector T cells and macrophages in colon tissues while increasing the number of regulatory T cells. Molecular docking analysis of the protein-ligand interaction profile revealed that the β5i-DPLG3 complex was more stable and efficient in the binding sites compared to those formed with ONX-0914 and LU-005i. Furthermore, DPLG3 reduced the protein levels of the canonical NF-κB p50 and p65, as well as the nuclear p65. Thus, DPLG3 constitutes a potentially efficacious clinical agent for autoimmune inflammatory diseases.
... The nature of the human immunoproteasome impairs its purification from endogenous sources, with a detrimental effect on its characterization and drug design and development. In one remarkable effort to overcome this limitation, a yeast strain was modified to incorporate in its 20S proteasomes chimeric yeast and human b5 subunits, designed to mimic the proteolytic activity of the endogenous human ib5 subunit while incorporating the yeast elements required for proteasome assembly and maturation (Huber et al., 2016). However, although these humanized proteasomes are already contributing to drug development, they are not physiological and only mimic ib5 without providing information regarding ligand binding at the ib1or ib2 active sites and no information regarding possible ligandinduced allosteric effects on the human immunoproteasome. ...
Article
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Proteasomes are essential in all eukaryotic cells. However, their function and regulation remain considerably elusive, particularly those of less abundant variants. We demonstrate the human 20S proteasome recombinant assembly and confirmed the recombinant complex integrity biochemically and with a 2.6 Å resolution cryo-EM map. To assess its competence to form higher-order assemblies, we prepared and analyzed recombinant human 20S-PA200, a poorly characterized nuclear complex. Its 3.0 Å resolution cryo-EM structure reveals the PA200 unique architecture; the details of its intricate interactions with the proteasome, resulting in unparalleled proteasome α ring rearrangements; and the molecular basis for PA200 allosteric modulation of the proteasome active sites. Non-protein cryo-EM densities could be assigned to PA200-bound inositol phosphates, and we speculate regarding their functional role. Here we open extensive opportunities to study the fundamental properties of the diverse and distinct eukaryotic proteasome variants and to improve proteasome targeting under different therapeutic conditions.
... The binding mode obtained from docking was compared to that of bortezomib [11], a covalent dipeptide boronic acid drug, and also to Ro19 [37], a nonpeptidic noncovalent inhibitor. The crystal structures of both compounds in complex with yeast 20S proteasome containing human β5i unit are available (bortezomib PDB code: 5L5F [38], Ro19 PDB code: 5M2B [16]). Ro19 and bortezomib bind differently and occupy quasi-orthogonal positions. ...
Article
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Large-scale virtual screening of boronic acid derivatives was performed to identify nonpeptidic covalent inhibitors of the β5i subunit of the immunoproteasome. A hierarchical virtual screening cascade including noncovalent and covalent docking steps was applied to a virtual library of over 104,000 compounds. Then, 32 virtual hits were selected, out of which five were experimentally confirmed. Biophysical and biochemical tests showed micromolar binding affinity and time-dependent inhibitory potency for two compounds. These results validate the computational protocol that allows the screening of large compound collections. One of the lead-like boronic acid derivatives identified as a covalent immunoproteasome inhibitor is a suitable starting point for chemical optimization.
... Peptide epoxyketones represent another class of β5i selective inhibitors (de Bruin et al., 2014;Dubiella et al., 2014;Fan et al., 2014;Groll, Korotkov, Huber, de Meijere, & Ludwig, 2015;Koroleva et al., 2015) amongst which PR-957, an analogue of carfilzomib, shows 20-to 40-fold selectivity towards β5i in MOLT-4 cells where both forms of the proteasome are expressed and showed evidence of disease reversal of rheumatoid arthritis mouse models (Muchamuel et al., 2009). PR-924, a selective inhibitor of the immunoproteasome with up to 250-fold selectivity towards β5i over β5c (Huber, Heinemeyer, de Bruin, Overkleeft, & Groll, 2016), selectively inhibited growth and triggered apoptosis in multiple myeloma cells over normal cells, validating the β5i site as a target for multiple myeloma treatment (Singh et al., 2011). These inhibitors are irreversible and covalently modify the active site threonine forming a protein-drug adduct. ...
Article
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Inhibitors of the proteasome have found broad therapeutic applications; however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non‐competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the β5i and β1i sites of the immunoproteasome over the β5c and β1c sites of the constitutive proteasome with nearly 20‐fold selective inhibition of β1i over the homologous β1c. Molecular modelling attributes the β1i over β1c selectivity to the small hydrophobic S1 pocket of β1i and β5i over β5c to site‐specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity.
Article
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Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone-H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast) marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful, however co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast, however this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell-cycle progression and kinetochore function in wild-type cells. Suppressors of CENP-A toxicity included gene deletions of subunits of three conserved chromatin-remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.
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Yeast and humans share thousands of genes despite a billion years of evolutionary divergence. While many human genes can functionally replace their yeast counterparts, nearly half of the tested shared genes cannot. For example, most yeast proteasome subunits are "humanizable", except subunits comprising the β-ring core, including β2c (HsPSMB7, a constitutive proteasome subunit). We developed a high-throughput pipeline to humanize yeast proteasomes by generating a large library of Hsβ2c mutants and screening them for complementation of a yeast β2 (ScPup1) knockout. Variants capable of replacing ScPup1 included (1) those impacting local protein-protein interactions (PPIs), with most affecting interactions between the β2c C-terminal tail and the adjacent β3 subunit, and (2) those affecting β2c proteolytic activity. Exchanging the full-length tail of human β2c with that of ScPup1 enabled complementation. Moreover, wild-type human β2c could replace yeast β2 if human β3 was also provided. Unexpectedly, yeast proteasomes bearing a catalytically inactive HsPSMB7-T44A variant that blocked precursor autoprocessing were viable, suggesting an intact propeptide stabilizes late assembly intermediates. In contrast, similar modifications in human β2i (HsPSMB10), an immuno-proteasome subunit and the co-ortholog of yeast β2, do not enable complementation in yeast, suggesting distinct interactions are involved in human immunoproteasome core assembly. Broadly, our data reveal roles for specific PPIs governing functional replaceability across vast evolutionary distances.
Article
A series of N, C-capped di- and tripeptides were designed as selective immunoproteasome inhibitors based on the known inhibitor 4-CA. Forty-nine new compounds were synthesized and evaluated, and the structure-activity relationship (SAR) of this compound class as β5i selective inhibitors were explored. Most of these compounds showed significant inhibition against the β5i subunit of the immunoproteasome and the most potent β5i inhibitor (15) showed an IC50 of 0.94 nM. A selective β5i inhibitor (54) with over 500-fold β5i/β5c selectivity was identified. Three of the inhibitors were found to selectively inhibit β5i and β5c, and showed no noticeable inhibition against the other four subunits. Six inhibitors with significant inhibitory activity against the HCT-116 cells were recognized, and the most active inhibitors, 14 and 50, showed IC50 values of 0.46 μM and 0.16 μM, respectively. Some selective β5i inhibitors exhibited significant inhibitory effects on the release of the cytokines TNF-α and IL-6. The results not only afford effective chemical tools to elucidate the relationships between subunit selectivity and pharmacological profiles, but also offer useful clues for further optimization and development of selective immunoproteasome inhibitors.
Article
A series of epoxyketone analogues with varying N-caps and P3-configurations were designed, synthesized and evaluated. We found that D-Ala in P3 was crucial for β5i selectivity over β5c. Notably, compounds 20j (β5i IC50 = 26.0 nM, 25-fold selectivity) and 20l (β5i IC50 = 25.1 nM, 24-fold selectivity) with the D-configuration at P3 were the most selective inhibitors. Although 20j and 20l showed only moderate anti-proliferative activity against RPMI-8226 and MM.1S cell lines, based on our experiments, it indicates that the inhibition of β5i alone is not sufficient to exert anticancer effects and may rely on the complementary inhibition of β1i, β5c and β5i. These data further increase our understanding of immunoproteasome inhibitors in hematologic malignancies.
Article
Proteasomes contribute to maintaining protein homeostasis and their inhibition is beneficial in certain types of cancer and in autoimmune diseases. However, the inhibition of the proteasomes in healthy cells leads to unwanted side-effects and significant effort has been made to identify inhibitors specific for the immunoproteasome, especially to treat diseases which manifest increased levels and activity of this proteasome isoform. Here, we report our efforts to discover fragment-sized inhibitors of the human immunoproteasome. The screening of an in-house library of structurally diverse fragments resulted in the identification of benzo[d]oxazole-2(3H)-thiones, benzo[d]thiazole-2(3H)-thiones, benzo[d]imidazole-2(3H)-thiones, and 1-methylbenzo[d]imidazole-2(3H)-thiones (with a general term benzoXazole-2(3H)-thiones) as inhibitors of the chymotrypsin-like (β5i) subunit of the immunoproteasome. A subsequent structure-activity relationship study provided us with an insight regarding growing vectors. Binding to the β5i subunit was shown and selectivity against the β5 subunit of the constitutive proteasome was determined. Thorough characterization of these compounds suggested that they inhibit the immunoproteasome by forming a disulfide bond with the Cys48 available specifically in the β5i active site. To obtain fragments with biologically more tractable covalent interaction, we performed a warhead scan, which yielded benzoXazole-2-carbonitriles as promising starting points for the development of selective immunoproteasome inhibitors with non-peptidic scaffolds.
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Accumulating evidence arising from numerous clinical studies indicate that assembled and functional 20S proteasome complexes circulate freely in plasma. Elevated levels of this core proteolytic complex have been found in the plasma of patients suffering from blood, skin and solid cancers, autoimmune disorders, trauma and sepsis. Moreover, in various diseases, there is a positive correlation between circulating 20S proteasome (c20S) levels and treatment efficacy and survival rates, suggesting the involvement of this under-studied c20S complex in pathophysiology. However, many aspects of this system remain enigmatic, as we still do not know the origin, biological role or mechanisms of extracellular transport and regulation of c20S proteasomes. In this review, we provide an overview of the current understanding of the c20S proteasome system and discuss the remaining gaps in knowledge.
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Target identification for biologically active small molecules remains a major barrier for drug discovery. Cancer cells exhibiting defective DNA mismatch repair (dMMR) have been used as a forward genetics system to uncover compound targets. However, this approach has been limited by the dearth of cancer cell lines that harbor naturally arising dMMR. Here, we establish a platform for forward genetic screening using CRISPR/Cas9 to engineer dMMR into mammalian cells. We demonstrate the utility of this approach to identify mechanisms of drug action in mouse and human cancer cell lines using in vitro selections against three cellular toxins. In each screen, compound-resistant alleles emerged in drug-resistant clones, supporting the notion that engineered dMMR enables forward genetic screening in mammalian cells.
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The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in off-label trials of Systemic Lupus Erythematosus. One potential mechanism of this clinical benefit is from the depletion of pathogenic immune cells (plasmablasts and plasmacytoid dendritic cells). However, bortezomib is cytotoxic against non-immune cells which limits its use for autoimmune diseases. An attractive alternative is to selectively inhibit the immune cell-specific immunoproteasome to deplete pathogenic immune cells and spare non-hematopoietic cells. Here we disclose the development of highly subunit-selective immunoproteasome inhibitors using insights obtained from the first bona fide human immunoproteasome co-crystal structures. Evaluation of these inhibitors revealed that immunoproteasome-specific inhibition does not lead to immune cell death as anticipated and that targeting viability requires inhibition of both immuno- and constitutive proteasomes. CRISPR/Cas9-mediated knock-out experiments confirmed upregulation of the constitutive proteasome upon disruption of the immunoproteasome, protecting cells from death. Thus, immunoproteasome inhibition alone is not a suitable approach to deplete immune cells.
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With the recent advances in biological structural electron microscopy (EM), protein structures can now be obtained by cryo-EM and single-particle analysis at resolutions that used to be achievable only by crystallographic or NMR methods. We have explored their application to study protein–ligand inter­actions using the human 20S proteasome, a well established target for cancer therapy that is also being investigated as a target for an increasing range of other medical conditions. The map of a ligand-bound human 20S proteasome served as a proof of principle that cryo-EM is emerging as a realistic approach for more general structural studies of protein–ligand interactions, with the potential benefits of extending such studies to complexes that are unfavourable to other methods and allowing structure determination under conditions that are closer to physiological, preserving ligand specificity towards closely related binding sites. Subsequently, the cryo-EM structure of the Plasmodium falciparum 20S proteasome, with a new prototype specific inhibitor bound, revealed the molecular basis for the ligand specificity towards the parasite complex, which provides a framework to guide the development of highly needed new-generation antimalarials. Here, the cryo-EM analysis of the ligand-bound human and P. falciparum 20S proteasomes is reviewed, and a complete description of the methods used for structure determination is provided, including the strategy to overcome the bias orientation of the human 20S proteasome on electron-microscope grids and details of the icr3d software used for three-dimensional reconstruction.
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Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a non-peptidic scaffold with the absence of an electrophile has been reported in a patent. Herein, we describe the mode of action of the lead compound using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the β5i substrate binding channel. Distinctive interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.
Article
The Mycobacterium tuberculosis (Mtb) 20S proteasome is vital for the pathogen to survive under nitrosative stress in vitro and to persist in mice. To qualify for drug development, inhibitors targeting Mtb 20S must spare both the human constitutive proteasome (c-20S) and immunoproteasome (i-20S). We recently reported members of a family of noncovalently binding dipeptide proteasome inhibitors that are highly potent and selective for Mtb 20S over human c-20S and i-20S. To understand the structural basis of their potency and selectivity, we have studied the structure-activity relationship of six derivatives and solved their co-crystal structures with Mtb 20S. The dipeptide inhibitors form an antiparallel -strand with the active site -strands. Selectivity is conferred by several features of Mtb 20S relative to its mouse counterparts, including a larger S1 pocket, additional hydrogen bonds in the S3 pocket, and hydrophobic interactions in the S4 pocket. Serine-20 and glutamine-22 of Mtb 20S interact with the dipeptides and confer Mtb-specific inhibition over c-20S and i-20S. The Mtb 20S and mammalian i-20S have a serine-27 that interacts strongly with the dipeptides, potentially explaining the higher inhibitory activity of the dipeptides toward i-20S over c-20S. This detailed structural knowledge will aid in optimizing the dipeptides as anti-tuberculosis drugs.
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Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.
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Despite the significant therapeutic advances achieved with proteasome inhibitors (PIs) such as bortezomib and carfilzomib in prolonging the survival of patients with multiple myeloma, the development of drug resistance, peripheral neuropathy, and pharmacokinetic limitations continue to pose major challenges when using these compounds. Ixazomib is a second-generation PI with improved activity over other PIs. Unlike bortezomib and carfilzomib, which are administered by injection, ixazomib is the first oral PI approved by US Food and Drug Administration. This review discusses the biochemical properties, mechanisms of action, preclinical efficacy, and clinical trial results leading to the US Food and Drug Administration approval of ixazomib.
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Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG35-55 and PLP139-151-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4(+) cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.
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MAIN is software that has been designed to interactively perform the complex tasks of macromolecular crystal structure determination and validation. Using MAIN, it is possible to perform density modification, manual and semi-automated or automated model building and rebuilding, real- and reciprocal-space structure optimization and refinement, map calculations and various types of molecular structure validation. The prompt availability of various analytical tools and the immediate visualization of molecular and map objects allow a user to efficiently progress towards the completed refined structure. The extraordinary depth perception of molecular objects in three dimensions that is provided by MAIN is achieved by the clarity and contrast of colours and the smooth rotation of the displayed objects. MAIN allows simultaneous work on several molecular models and various crystal forms. The strength of MAIN lies in its manipulation of averaged density maps and molecular models when noncrystallographic symmetry (NCS) is present. Using MAIN, it is possible to optimize NCS parameters and envelopes and to refine the structure in single or multiple crystal forms.
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Purpose: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR). Experimental Design: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors. Results: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure. Conclusion: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile. Clin Cancer Res; 17(9); 2734–43. ©2011 AACR.
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The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based [corrected] on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S beta5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.
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Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are ‘discoverable’ through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications.
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When cells are stimulated with pro-inflammatory cytokines, most of their constitutively expressed proteasomes are replaced with immunoproteasomes, which increase the production of peptides for presentation on MHC class I molecules. In addition, cortical thymic epithelial cells selectively express a type of proteasome known as the thymoproteasome that is required for the positive selection of thymocytes. Here, we discuss how these specialized types of proteasome shape the T cell receptor repertoire of cytotoxic T lymphocytes and propose that immunoproteasomes have functions, in addition to antigen processing, that influence cytokine production and T cell differentiation, survival and function. We also discuss how inhibitors of immunoproteasomes can suppress undesired T cell responses in autoimmune diseases.
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The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low-molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I-restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-gamma and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.
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In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.
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The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (alpha1...alpha7, beta1...beta7)2 complex in four stacked rings and occupy unique locations. The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances. The beta-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle. The proforms of three of the seven different beta-type subunits, beta1/PRE3, beta2/PUP1 and beta5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1. These three beta-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity. Other beta-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.
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The proteasome is a large protease complex consisting of multiple catalytic subunits that function simultaneously to digest protein substrates. This complexity has made deciphering the role each subunit plays in the generation of specific protein fragments difficult. Positional scanning libraries of peptide vinyl sulfones were generated in which the amino acid located directly at the site of hydrolysis (P1 residue) was held constant and sequences distal to that residue (P2, P3, and P4 positions) were varied across all natural amino acids (except cysteine and methionine). Binding information for each of the individual catalytic subunits was obtained for each library under a variety of different conditions. The resulting specificity profiles indicated that substrate positions distal to P1 are critical for directing substrates to active subunits in the complex. Furthermore, specificity profiles of IFN-gamma-regulated subunits closely matched those of their noninducible counterparts, suggesting that subunit swapping may modulate substrate processing by a mechanism that does require a change in the primary sequence specificity of individual catalytic subunits in the complex. Finally, specificity profiles were used to design specific inhibitors of a single active site in the complex. These reagents can be used to further establish the role of each subunit in substrate processing by the proteasome.
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One of the most important aspects of macromolecular structure refinement is the use of prior chemical knowledge. Bond lengths, bond angles and other chemical properties are used in restrained refinement as subsidiary conditions. This contribution describes the organization and some aspects of the use of the flexible and human/machine-readable dictionary of prior chemical knowledge used by the maximum-likelihood macromolecular-refinement program REFMAC5. The dictionary stores information about monomers which represent the constitutive building blocks of biological macromolecules (amino acids, nucleic acids and saccharides) and about numerous organic/inorganic compounds commonly found in macromolecular crystallography. It also describes the modifications the building blocks undergo as a result of chemical reactions and the links required for polymer formation. More than 2000 monomer entries, 100 modification entries and 200 link entries are currently available. Algorithms and tools for updating and adding new entries to the dictionary have also been developed and are presented here. In many cases, the REFMAC5 dictionary allows entirely automatic generation of restraints within REFMAC5 refinement runs.
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Proteasomes are responsible for generating peptides presented by the class I major histocompatibility complex (MHC) molecules of the immune system. Here, we report the identification of a previously unrecognized catalytic subunit called β5t. β5t is expressed exclusively in cortical thymic epithelial cells, which are responsible for the positive selection of developing thymocytes. Although the chymotrypsin-like activity of proteasomes is considered to be important for the production of peptides with high affinities for MHC class I clefts, incorporation of β5t into proteasomes in place of β5 or β5i selectively reduces this activity. We also found that β5t-deficient mice displayed defective development of CD8+ T cells in the thymus. Our results suggest a key role for β5t in generating the MHC class I–restricted CD8+ T cell repertoire during thymic selection.
Article
This work reports the development of highly potent and selective inhibitors of the β5c catalytic activity of human constitutive proteasomes. The work details the design principles - large hydrophobic P3 residue, small hydrophobic P1 residue - that led to the synthesis of a panel of peptide epoxyketones; their evaluation and the selection of the most promising compounds for further analyses. Structure-activity-relationships detail how in a logical order the β1c/i, β2c/i and β5i activities became resistant to inhibition as compounds were diversified stepwise. The most effective compounds were obtained as a mixture of cis- and trans-biscyclohexyl isomers and enantioselective synthesis resolved this issue. Studies on yeast proteasome structures complexed to some of the compounds provide a rationale for the potency and specificity. Substitution of the N-terminus in the most potent compound for a more soluble equivalent led to a cell-permeable molecule that selectively and completely blocks β5c in cells expressing both constitutive proteasomes and immunoproteasomes.
Article
The immunoproteasome is predominantly expressed in monocytes and lymphocytes and is responsible for the generation of antigenic peptides for cell-mediated immunity. Upon the exposure of inflammatory cytokines IFN-γ and TNF-α, constitutive subunits can be replaced by the synthesis of the imrmmo-eore particles βli, β2i and β5i. Recent studies demonstrated that the immunoproteasome function is not only limited to MHC class I presentation, but it is also implicated in a number of pathological disorders including hematological malignancies, inflammatory and autoimmune diseases. At present the commercially available proteasome inhibitors Bortezomib and Carfilzomib, which have been validated in multiple myeloma and other diseases, appear to target both the constitutive and tmmuno-proteasomes indiscriminately. This lack of specificity may, in part, explain some of the side effects of these agents. In contrast, by selectively targeting the immunoproteasome, it may be possible to keep the antimye-loma and antilymphoma efficacy unchanged and, at the same time, to increase the therapeutic index. The aim of this review article is to discuss the most promising immunoproteasome core particle-selective inhibitors which have been developed in the recent years, with a particular attention to their structural features, mechanism of action and therapeutic application.
Article
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.
Article
Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α',β'epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts: 1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. 2) Identification of the β2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between β2c and β2i. 3) The compound Ac-LAA-ep was found to favor subunit β5c over β5i by three orders of magnitude. 4) Yeast β1 and human β1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. 5) Exceptional structural features in the β1/2 substrate binding channel give rise to the β1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts towards subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders.
Article
Inhibition of the 20S proteasome by bortezomib (Velcade) constitutes a successfully applied therapy for blood cancer. However, emerging resistance restricts its medicinal use. For example, mutations in the proteolytically active β5-subunit of the proteasome, the main target of inhibitors, were reported to impair drug binding and thus to reduce therapeutic efficacy. Using yeast as a model system, we describe here a systematic evaluation of these mutations by cell growth analysis, proteasome inhibition assays, and X-ray crystallography. The 11 mutants examined display decreased proliferation rates, impaired proteolytic activity, and marked resistance to bortezomib as well as the α',β'-epoxyketone inhibitors carfilzomib (Kyprolis) and ONX 0914, while the second-generation compound carfilzomib was the least affected. In total, 49 proteasome X-ray structures, including structural data on proteasome-carfilzomib complexes, reveal three distinct molecular mechanisms that hamper both drug binding and natural substrate turnover to an extent that is still compatible with cell survival. Copyright © 2015 Elsevier Ltd. All rights reserved.
Article
Proteasome inhibition is highly effective as a treatment for multiple myeloma, and recently carfilzomib was granted US FDA approval for the treatment of relapsed and refractory multiple myeloma. Here, we report the X-ray crystal structure of the human constitutive 20S proteasome with and without carfilzomib bound at 2.9 and 2.6 Å, respectively. Our data indicate that the S3 and S4 binding pockets play a pivotal role in carfilzomib's selectivity for chymotrypsin-like sites. Structural comparison with the mouse immunoproteasome crystal structure reveals amino acid substitutions that explain carfilzomib's slight preference for chymotrypsin-like subunits of constitutive proteasomes. In addition, comparison of the human proteasome:carfilzomib complex with the mouse proteasome:PR-957 complex reveals new details that explain why PR-957 is selective for immunoproteasomes. Together, the data presented here support the design of inhibitors for either constitutive or immunoproteasomes, with implications for the treatment of cancers as well as autoimmune and neurodegenerative diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.
Article
Mammalian genomes encode seven catalytic proteasome subunits, namely β1c, β2c, β5c (assembled into constitutive 20S proteasome core particles), β1i, β2i, β5i (incorporated into immunoproteasomes) and the thymoproteasome-specific subunit, β5t. Extensive research in the past decades has yielded numerous potent proteasome inhibitors including compounds currently used in the clinic to treat multiple myeloma and mantle cell lymphoma. Proteasome inhibitors that selectively target combinations of β1c/β1i, β2c/β2i or β5c/β5i are available, yet ligands truly selective for a single proteasome activity are scarce. In this work we report the development of cell-permeable β1i as well as β5i selective inhibitors, which outperform existing leads in terms of selectivity and/or potency. These compounds are the result of a rational design strategy using known inhibitors as starting points and introducing structural features according to the X-ray structures of the murine constitutive and immunoproteasome 20S core particles.
Article
PR-924 is a novel prototypic immunoproteasome inhibitor bearing markedly enhanced specificity for the β5i immunoproteasome subunit, compared to the classical proteasome inhibitor bortezomib. Here, we assessed the growth inhibitory potential of PR-924 in three human hematologic malignancy cell lines (CCRF-CEM, THP1, and 8226) and their bortezomib-resistant sublines. Parental cells displayed equal sensitivity to PR-924 (IC50: 1.5-2.8 μM), whereas their bortezomib-resistant tumor lines displayed a 10-12 fold cross-resistance to PR-924. However, PR-924 cross-resistance factors for bortezomib-resistant sublines were markedly lower compared to the resistance factors to bortezomib. Proteasome inhibition experiments confirmed that PR-924 specifically inhibited β5i activity, even far below concentrations that exerted anti-proliferative activity. We further determined whether PR-924 activity might be compromised by acquisition of drug resistance phenomena. Indeed, CEM cells rendered stepwise resistant to 20 μM PR-924 (CEM/PR20) displayed 13-fold PR-924-resistance and 10-fold cross-resistance to bortezomib. CEM/PR20 cells were devoid of mutations in the PSMB8 gene (encoding β5i), but acquired Met45Ile mutation in the PSMB5 gene (encoding constitutive β5), consistent with β5 mutations observed in bortezomib-resistant cells. Furthermore, compared to parental CEM cells, CEM/PR20 cells exhibited 2.5-fold upregulation of constitutive proteasome subunit expression, whereas immunoproteasome subunit expression was 2-fold decreased. In conclusion, PR-924 displayed potent anti-leukemic activity including toward bortezomib-resistant leukemia cells. Despite the specificity of PR-924 to the β5i immunoproteasome subunit, its anti-leukemic effect required concentrations that blocked both β5 and β5i subunits. This is underscored by the emergence of mutations in PSMB5 rather than in PSMB8.
Article
Probing the unknown: The immunoproteasome, an alternative form of the constitutive proteasome, has been implicated in a number of pathological states such as cancer and autoimmune diseases. In an effort to understand the role of the immunoproteasome in cells, the first immunoproteasome-specific near-infrared fluorescent probe has been developed.
Article
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.
Article
The combination of X-ray crystallography and kinetic studies of proteasome:ligand complexes has proven to be an important tool in inhibitor analysis of this crucial protein degradation machinery. Here, we describe in detail the purification protocols, proteolytic activity assays, crystallisation methods, and structure determination for the yeast 20S proteasome (CP) in complex with its inhibitors. The fusion of these advanced techniques offers the opportunity to further optimise drugs which are already tested in different clinical phase studies, as well as to design new promising proteasome lead structures which might be suitable for their application in medicine, plant protection, and antibiotics.
Article
Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit β5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of β5c/X but not β5i, thereby explaining the selectivity of PR-957 for β5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.
Article
To investigate the hypothesis that proteasome inhibition may have potential in the treatment of SLE, by targeting plasmacytoid dendritic cells (PDCs) and plasma cells, both of which are critical in disease pathogenesis. Lupus-prone mice were treated with the nonselective proteasome inhibitors carfilzomib and bortezomib, the immunoproteasome inhibitor ONX 0914, or vehicle control. Tissue was harvested and analyzed by flow cytometry using standard markers. Nephritis was monitored by evaluation for proteinuria and by histologic analysis of kidneys. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay (ELISA), and total IgG and dsDNA antibody-secreting cells (ASCs) by enzyme-linked immunospot assay. Human peripheral blood mononuclear cells or mouse bone marrow cells were incubated with Toll-like receptor (TLR) agonists and proteasome inhibitors, and interferon-α (IFNα) levels were measured by ELISA and flow cytometry. Early treatment of lupus-prone mice with the dual-targeting proteasome inhibitors carfilzomib or bortezomib or the immunoproteasome-specific inhibitor ONX 0914 prevented disease progression, and treatment of mice with established disease dramatically abrogated nephritis. Treatment had profound effects on plasma cells, with greater reductions in autoreactive than in total IgG ASCs, an effect that became more pronounced with prolonged treatment and was reflected in decreasing serum autoantibody levels. Notably, proteasome inhibition efficiently suppressed production of IFNα by TLR-activated PDCs in vitro and in vivo, an effect mediated by inhibition of both PDC survival and PDC function. Inhibition of the immunoproteasome is equally efficacious as dual targeting agents in preventing lupus disease progression by targeting 2 critical pathways in disease pathogenesis, type I IFN activation and autoantibody production by plasma cells.
Article
PR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase-8, caspase-9, BID, PARP and cytochrome-c release. In vivo administration of PR-924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM.
Article
The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic determination of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the number of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.
Article
Proteasomes degrade most proteins in mammalian cells and are established targets of anticancer drugs. All eukaryotic proteasomes have three types of active sites: chymotrypsin-like, trypsin-like, and caspase-like. Chymotrypsin-like sites are the most important in protein degradation and are the primary target of most proteasome inhibitors. The biological roles of trypsin-like and caspase-like sites and their potential as cotargets of antineoplastic agents are not well defined. Here we describe the development of site-specific inhibitors and active-site probes of chymotrypsin-like and caspase-like sites. Using these compounds, we show that cytotoxicity of proteasome inhibitors does not correlate with inhibition of chymotrypsin-like sites and that coinhibition of either trypsin-like and/or caspase-like sites is needed to achieve maximal cytotoxicity. Thus, caspase-like and trypsin-like sites must be considered as cotargets of anticancer drugs.
Article
Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits beta5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138+ tumor cells. Although specific inhibition of either LMP7 or beta5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both beta5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2alpha suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors.
Article
We have cloned and analysed the second mouse MHC-linked proteasome subunit, designated MC13, which appears to be homologous to the human RING10 proteasome protein. The isolated cDNA has an ORF encoding a protein of 276 amino acids with a molecular weight of ca. 30 kDa. Sequence alignment reveals that the subunit MC13 and several other mammalian proteasome subunits are encoded by a second proteasome gene family. This second gene family encodes subunits of the beta-type, reveals striking sequence similarities with the beta-subunit of archaebacterial proteasomes and is related to, but distinct from, the genes encoding the so-called alpha-type subunits.
Article
The three-dimensional structure of the proteasome from the archaebacterium Thermoplasma acidophilum has been elucidated by x-ray crystallographic analysis by means of isomorphous replacement and cyclic averaging. The atomic model was built and refined to a crystallographic R factor of 22.1 percent. The 673-kilodalton protease complex consists of 14 copies of two different subunits, alpha and beta, forming a barrel-shaped structure of four stacked rings. The two inner rings consist of seven beta subunits each, and the two outer rings consist of seven alpha subunits each. A narrow channel controls access to the three inner compartments. The alpha 7 beta 7 beta 7 alpha 7 subunit assembly has 72-point group symmetry. The structures of the alpha and beta subunits are similar, consisting of a core of two antiparallel beta sheets that is flanked by alpha helices on both sides. The binding of a peptide aldehyde inhibitor marks the active site in the central cavity at the amino termini of the beta subunits and suggests a novel proteolytic mechanism.
Article
Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.
Article
The 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.
Article
Intracellular protein degradation is one of the most precisely regulated processes in living cells. The main component of the degradation machinery is the 20S proteasome present in eukaryotes as well as in prokaryotes. We have developed successful purification protocols for the 20S proteasome in its native state using an affinity tag strategy. This chapter describes in detail the purification protocols, proteolytic activity assays, crystallization, and structure determination for the yeast 20S proteasome. The crystal structure of the eukaryotic proteasome opens new possibilities for identifying, characterizing, and elucidating the mode of action for natural and synthetic inhibitors, which affect its function. Some of these compounds may find therapeutic applications in contemporary medicine.
Article
The dipeptide boronic acid bortezomib, also termed VELCADE, is a proteasome inhibitor now in use for the treatment of multiple myeloma, and its use for the treatment of other malignancies is being explored. We determined the crystal structure of the yeast 20S proteasome in complex with bortezomib to establish the specificity and binding mode of bortezomib to the proteasome's different catalytically active sites. This structure should enable the rational design of new boronic acid derivatives with improved affinities and specificities for individual active subunits.
Article
Clinical studies with bortezomib have validated the proteasome as a therapeutic target for the treatment of multiple myeloma and non-Hodgkin's lymphoma. However, significant toxicities have restricted the intensity of bortezomib dosing. Here we describe the antitumor activity of PR-171, a novel epoxyketone-based irreversible proteasome inhibitor that is currently in clinical development. In comparison to bortezomib, PR-171 exhibits equal potency but greater selectivity for the chymotrypsin-like activity of the proteasome. In cell culture, PR-171 is more cytotoxic than bortezomib following brief treatments that mimic the in vivo pharmacokinetics of both molecules. Hematologic tumor cells exhibit the greatest sensitivity to brief exposure, whereas solid tumor cells and nontransformed cell types are less sensitive to such treatments. Cellular consequences of PR-171 treatment include the accumulation of proteasome substrates and induction of cell cycle arrest and/or apoptosis. Administration of PR-171 to animals results in the dose-dependent inhibition of the chymotrypsin-like proteasome activity in all tissues examined with the exception of the brain. PR-171 is well tolerated when administered for either 2 or 5 consecutive days at doses resulting in >80% proteasome inhibition in blood and most tissues. In human tumor xenograft models, PR-171 mediates an antitumor response that is both dose and schedule dependent. The antitumor efficacy of PR-171 delivered on 2 consecutive days is stronger than that of bortezomib administered on its clinical dosing schedule. These studies show the tolerability, efficacy, and dosing flexibility of PR-171 and provide validation for the clinical testing of PR-171 in the treatment of hematologic malignancies using dose-intensive schedules.