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Proteasome inhibitors are one of the most important classes of agents to have emerged for the treatment of multiple myeloma in the past two decades, and now form one of the backbones of treatment. Three agents in this class have been approved by the United States Food and Drug Administration—the first-in-class compound bortezomib, the second-generation agent carfilzomib, and the first oral proteasome inhibitor, ixazomib. The success of this class of agents is due to the exquisite sensitivity of myeloma cells to the inhibition of the 26S proteasome, which plays a critical role in the pathogenesis and proliferation of the disease. Proteasome inhibition results in multiple downstream effects, including the inhibition of NF-κB signaling, the accumulation of misfolded and unfolded proteins, resulting in endoplasmic reticulum stress and leading to the unfolded protein response, the downregulation of growth factor receptors, suppression of adhesion molecule expression, and inhibition of angiogenesis; resistance to proteasome inhibition may arise through cellular responses mediating these downstream effects. These multiple biologic consequences of proteasome inhibition result in synergistic or additive activity with other chemotherapeutic and targeted agents for myeloma, and proteasome inhibitor-based combination regimens have become established as a cornerstone of therapy throughout the myeloma treatment algorithm, incorporating agents from the other key classes of antimyeloma agents, including the immunomodulatory drugs, monoclonal antibodies, and histone deacetylase inhibitors. This review gives an overview of the critical role of the proteasome in myeloma and the characteristics of the different proteasome inhibitors and provides a comprehensive summary of key clinical efficacy and safety data with the currently approved proteasome inhibitors.

Structural and functional characterizations show that the specificity of the Plasmodium falciparum proteasome is sufficiently unique from that of the human proteasome to allow selective targeting with inhibitors. Plasmodium -specific proteasome inhibitors Compounds that target the Plasmodium proteasome are potentially valuable antimalarial therapeutics, but most proteasome inhibitors so far tested are also toxic for the human proteasome. In this study, the authors used a substrate profiling method to define amino acid preferences specific to the parasite proteasome and used chemical synthesis to produce inhibitors that preferentially target the β2 subunit. They demonstrate that such an inhibitor attenuates parasite growth in vivo and synergizes with artemisinin drugs to potentiate growth inhibition. A cryo-EM structure of the P. falciparum 20S proteasome bound to the inhibitor provides valuable molecular insight that can be used to further refine inhibitor design. The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation 1 . Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle 2 , 3 , 4 , 5 . Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents 2 , 6 . Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials 7 , 8 , we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

The ability of ubiquitin to form up to eight different polyubiquitin chain linkages generates complexity within the ubiquitin proteasome system, and accounts for the diverse roles of ubiquitination within the cell. Understanding how each type of ubiquitin linkage is correctly interpreted by ubiquitin binding proteins provides important insights into the link between chain recognition and cellular fate. A major function of ubiquitination is to signal degradation of intracellular proteins by the 26S proteasome. Lysine-48 (K48) linked polyubiquitin chains are well established as the canonical signal for proteasomal degradation, but recent studies show a role for other ubiquitin linked chains in facilitating degradation by the 26S proteasome. Here, we review how different types of polyubiquitin linkage bind to ubiquitin receptors on the 26S proteasome, how they signal degradation and discuss the implications of ubiquitin chain linkage in regulating protein breakdown by the proteasome.

Hyer et al . generate a potent and specific small-molecule inhibitor of the E1 ubiquitin-activating enzyme UBE1 that has antitumor activity in mice against a wide variety of tumor types. The ubiquitin–proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Background Loss of P-glycoprotein (P-gp) at the blood–brain barrier contributes to amyloid-β (Aβ) brain accumulation in Alzheimer’s disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aβ triggers P-gp loss by activating the ubiquitin–proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aβ accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade ® ; BTZ), protects blood–brain barrier P-gp from degradation in hAPP mice in vivo. Methods We treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood–brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aβ plasma and brain levels with ELISA. Results We found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood–brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aβ40 and Aβ42 brain levels compared to vehicle-treated hAPP mice. Conclusions Our data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aβ brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aβ transport across the blood–brain barrier may prove an effective treatment for patients.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi. Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the β5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the β4 and β5 proteasome subunits. This induced pocket exploits β4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

A selective inhibitor of the kinetoplastid proteasome (GNF6702) is identified that is highly efficacious in vivo , clearing the parasites that cause leishmaniasis, Chagas disease and sleeping sickness from mice, highlighting the possibility of developing a single class of drugs for these neglected diseases. Three tropical diseases targeted by new drug Chagas disease, leishmaniasis, and sleeping sickness are caused by the kinetoplastid parasites Trypanosoma cruzi , Leishmania spp. and Trypanosoma brucei spp., respectively, and affect 20 million people worldwide. This study reports the results of a screen to find new conserved molecular targets and broad spectrum drugs that could be used to treat all three diseases. A selective inhibitor of the kinetoplastid proteasome (GNF6702) was identified as the most effective. It is highly efficacious in vivo , clearing parasites from mice in all three models of infection. GNF6702 is a non-competitive inhibitor, specific for kinetoplastid proteasome, and is well-tolerated in mice. These results highlight the possibility of developing a single class of drugs for these neglected diseases. Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually 1 . The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi , Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target 2 . However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

The ubiquitin-proteasome system (UPS) degrades intracellular proteins into peptide fragments that can be presented by major histocompatibility complex (MHC) class I molecules. While the UPS is functional in all mammalian cells, its subunit composition differs depending on cell type and stimuli received. Thus, cells of the hematopoietic lineage and cells exposed to (pro)inflammatory cytokines express three proteasome immunosubunits, which form the catalytic centers of immunoproteasomes, and the proteasome activator PA28. Cortical thymic epithelial cells express a thymus-specific proteasome subunit that induces the assembly of thymoproteasomes. We here review new developments regarding the role of these different proteasome components in MHC class I antigen processing, T cell repertoire selection and CD8 T cell responses. We further discuss recently discovered functions of proteasomes in peptide splicing, lymphocyte survival and the regulation of cytokine production and inflammatory responses.

PA28 (also known as 11S, REG or PSME) is a family of proteasome regulators whose members are widely present in many of the eukaryotic supergroups. In jawed vertebrates they are represented by three paralogs, PA28α, PA28β, and PA28γ, which assemble as heptameric hetero (PA28αβ) or homo (PA28γ) rings on one or both extremities of the 20S proteasome cylindrical structure. While they share high sequence and structural similarities, the three isoforms significantly differ in terms of their biochemical and biological properties. In fact, PA28α and PA28β seem to have appeared more recently and to have evolved very rapidly to perform new functions that are specifically aimed at optimizing the process of MHC class I antigen presentation. In line with this, PA28αβ favors release of peptide products by proteasomes and is particularly suited to support adaptive immune responses without, however, affecting hydrolysis rates of protein substrates. On the contrary, PA28γ seems to be a slow-evolving gene that is most similar to the common ancestor of the PA28 activators family, and very likely retains its original functions. Notably, PA28γ has a prevalent nuclear localization and is involved in the regulation of several essential cellular processes including cell growth and proliferation, apoptosis, chromatin structure and organization, and response to DNA damage. In striking contrast with the activity of PA28αβ, most of these diverse biological functions of PA28γ seem to depend on its ability to markedly enhance degradation rates of regulatory protein by 20S proteasome. The present review will focus on the molecular mechanisms and biochemical properties of PA28γ, which are likely to account for its various and complex biological functions and highlight the common features with the PA28αβ paralog.