Proteasome structure, function, and lessons learned from beta-lactone inhibitors

The 26S proteasome is the enzymatic core engine of the ubiquitin and proteasome dependent proteolytic system (UPS), the major eukaryotic pathway for regulated protein degradation. The UPS plays a pivotal role in cellular protein turnover, protein quality control, antigen processing, signal transduction, cell cycle regulation, cell differentiation and apoptosis, inspiring in-depth studies of proteasome structure and function and the search for selective inhibitors. Structural studies revealed that the 26S proteasome comprises up to two 19S regulatory caps flanking a cylindrical 20S core particle, which houses the proteolytic subunits and is present in all kingdoms of life. This review highlights current understanding of 20S architecture, maturation and assembly, the mechanism for selective degradation of protein substrates targeted for destruction, and relationships to other proteases. This knowledge base has benefited from structurally diverse proteasome inhibitors discovered from unique sources, including terrestrial and marine actinomycetes that produce the β-lactone-γ- lactam superfamily of inhibitors, including omuralide, salinosporamide A (marizomib; NPI-0052) and the cinnabaramides. These "minimalist inhibitors" utilize dense functionality to maximum efficiency for potent and selective proteasome inhibition and have advanced from biochemical tools to potential agrochemicals and anticancer agents. In this review, lessons learned from the β-lactone-γ-lactam superfamily are presented, with an emphasis on their unique binding mechanisms elucidated through structural biology in concert with medicinal chemistry. Distinctions between slowly reversible and irreversible inhibitors are discussed, together with the relationship of irreversible binding at the molecular level to prolonged duration proteasome inhibition in tumor cells, and in vitro and in vivo efficacy.     

Targeting the ubiquitin-proteasome pathway: an emerging concept in cancer therapy

Selective degradation of proteins by the ubiquitin-proteasome pathway is a critical determinant for maintaining cellular homeostasis. Most intracellular proteins are degraded by the proteasome, a multicatalytic enzyme complex containing a 20S catalytic core and two 19S regulatory complexes. Many proteasome target proteins are involved in the regulation of important processes of carcinogenesis and cancer cell survival, such as cell cycle progression, cell proliferation, differentiation and apoptosis. Indeed, the ubiquitin-proteasome-dependent degradation pathway plays an essential role in both the up-regulation of cell proliferation and down-regulation of cell death in human cancer cells. Both in vitro and in vivo experimental and clinical results have demonstrated the potential use of proteasome inhibitors as novel anticancer drugs. Proteasome inhibition in cancer cells leads to accumulation of pro-apoptotic target proteins followed by induction of cell death. The clinical efficacy of the proteasome inhibitor bortezomib toward multiple myeloma and other hematologic malignancies provides the "proof of concept" that targeting the proteasome is a promising strategy for cancer treatment. Several other proteasome inhibitors have also been identified from natural resources, such as marine microbial metabolites, green tea polyphenols, flavonoids, and medicinal compounds. Additionally, the use of metal complexes as proteasome inhibitors has also been investigated as a potential anticancer strategy. The clinical significance of targeting the tumor survival-associated proteasome pathway for cancer treatment, intervention and prevention will be discussed.     

三肽四氮唑类20S蛋白酶体抑制剂的设计、合成与活性研究

泛素-蛋白酶体途径是细胞内降解蛋白质的一种主要方式,由20S蛋白酶体来完成蛋白质的降解。本文在已经报道的肽类抑制剂的基础上,设计合成了一类三肽四氮唑化合物,通过1H NMR、MS以及元素分析对化合物结构进行了表征。活性评价结果表明,有3个目标化合物(6b、6d和6h)具有较好的抑制20S蛋白酶体类胰凝乳蛋白酶的活性。分子对接研究显示,这类新型C端基肽类化合物能通过与活性位点非共价相互作用而与蛋白酶体结合。     

A patent review of the ubiquitin ligase system: 2015–2018

ABSTRACT

Introduction: Ubiquitin-proteasome system (UPS) has been validated as a novel anticancer drug target in the past 20 years. The UPS contains two distinct steps: ubiquitination of a substrate protein by ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3), and substrate degradation by the 26S proteasome complex. The E3 enzyme is the central player in the ubiquitination step and has a wide range of specific substrates in cancer cells, offering great opportunities for discovery and development of selective drugs.

Areas covered: This review summarizes the recent advances in small molecule inhibitors of E1s, E2s, and E3s, with a focus on the latest patents (from 2015 to 2018) of E3 inhibitors and modulators.

Expert opinion: One strategy to overcome limitations of current 20S proteasome inhibitors is to discover inhibitors of the upstream key components of the UPS, such as E3 enzymes. E3s play important roles in cancer development and determine the specificity of substrate ubiquitination, offering novel target opportunities. E3 modulators could be developed by rational design, natural compound or library screening, old drug repurposes, and application of other novel technologies. Further understanding of mechanisms of E3–substrate interaction will be essential for discovering and developing next-generation E3 inhibitors as effective anticancer drugs.     

A new therapy with bortezomib, an oncologic medicinal product of the year 2004

Proteasome-mediated proteolysis is a mechanism for mediating important regulatory proteins within the cell. Proteins that have been targeted for degradation by the proteasome are convalently tagged with a poly-ubiquitin protein chain prior to be recognized by the 19S subunit of proteasome. This degradation system controls the expression of a wide variety of cellular targets including tumor suppressors such as p53, inhibitor of nuclear factor NFkappaB, cyclin-dependent kinase inhibitors such as p21 and p27. Because of these functions, the proteasome has become a new target for cancer treatment. The potent and selective proteasome inhibitor, PS-341 or Velcade was approved in the United States and launched in may 2003 for the treatment of multiple myeloma patients who have received at least two prior therapies. On April 2004, the European commission granted marketing authorization for Velcade with the same indication. The same year 2004, the Nobel Prize in chemistry was awarded to three researchers "for the discovery of ubitiquin-mediated protein degradation", a regulated process by which proteins are cleaved into peptides inside cells.     

Proteasome inhibition as a new therapeutic principle in hematological malignancies

The intracellular concentration of proteins in both normal and tumor cells are regulated by the balance between the rates of protein synthesis vs. degradation. The ubiquitin-proteasome pathway is the main intracellular cascade for controlled degradation of proteins and has attracted in recent years major interest not only because of its biochemical complexity and the intricate regulation of its function, but also because diverse cell cycle regulators and modulators of apoptosis are subject to regulation by proteasome function, and can therefore be significantly affected by small molecule inhibitors of the proteolytic activity of the proteasome. In fact, bortezomib, the prototypic member of this class of agents, was recently approved by the U.S. Food and Drug Administration for the treatment of advanced multiple myeloma patients. This review article focuses on the exciting recent progress in the use of proteasome inhibitors, with emphasis on the bench-to-bedside research effort which provided the foundation for clinical development of bortezomib for the treatment of multiple myeloma, as well as other hematologic malignancies, such as mantle cell lymphoma.     

Targeting the proteasome pathway

Background: The ubiquitin–proteasome pathway functions as a main pathway in intracellular protein degradation and plays a vital role in almost all cellular events. Various inhibitors of this pathway have been developed for research purposes. The recent approval of bortezomib (PS-341, Velcade^®), a proteasome inhibitor, for the treatment of multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and other components of the ubiquitin–proteasome pathway. Objectives: We review the current understanding of the ubiquitin–proteasome pathway and inhibitors targeting this pathway, including proteasome inhibitors, as candidate drugs for chemical therapy. Methods: Preclinical and clinical data for inhibitors of the proteasome and the ubiquitin–proteasome pathway are discussed. Conclusions: The proteasome and other members in the ubiquitin–proteasome pathway have emerged as novel therapeutic targets.     

Proteasome inhibitors as therapeutic agents: current and future strategies

In cells, protein degradation is a key pathway for the destruction of abnormal or damaged proteins as well as for the elimination of proteins whose presence is no longer required. Among the various cell proteases, the proteasome, a multicatalytic macromolecular complex, is specifically required for the degradation of ubiquitinated proteins. In normal cells, the proteasome ensures the elimination of numerous proteins that play critical roles in cell functions throughout the cell cycle. Defects in the activity of this proteolytic machinery can lead to the disorders of cell function that is believed to be the root cause of certain diseases. Indeed, many proteins involved in the control of cell cycle transitions are readily destroyed by the proteasome once their tasks have been accomplished. Moreover, because proteasome inhibitors can provoke cell death, it has been suggested that proteasomes must be continually degrading certain apoptotic factors. For these reasons, proteasome inhibition has become a new and potentially significant strategy for the drug development in cancer treatment. The proteasome possesses three major peptidase activities that can individually be targeted by drugs. Different classes of proteasome inhibitors are reviewed here. In addition, we present new pseudopeptides with the enriched nitrogen backbones bearing a side chain and a modified C-terminal position that inhibit proteasome activity.     

磺胺类黄酮衍生物20S蛋白酶体抑制剂的设计、合成与活性评价

本文通过计算机辅助设计,设计并合成了一系列磺胺类黄酮衍生物作为非共价20S蛋白酶体抑制剂,并对其生物活性进行了测试。与先导化合物相比（β5亚基的IC50值为14.0μM）,化合物仅表现出局部的改善,但仍可作为一类潜在的20S蛋白酶体抑制剂。     

Hsp90β inhibitors prevent GLT-1 degradation but have no beneficial efficacy on absence epilepsy

The loss of glutamate transporter-1 (GLT-1) is associated with temporal lobe epilepsy (TLE). A recent study reported that Hsp90β interacted with GLT-1 and recruited it to 20S proteasome for degradation. Therefore, inhibiting Hsp90β may be a new strategy for treating epilepsy. So far, no studies have shown whether the inhibition of Hsp90β had therapeutic effects on absence epilepsy. Using a model of absence epilepsy, we demonstrated that 17-allylamino-17-demethoxygeldanamycin (17AAG) and Ganetespib (STA9090) had no therapeutic effect. Although this is a negative result, it also has a meaningful exploration value for whether Hsp90 inhibitors have therapeutic effects on other epilepsy types.     

Proteasomes as drug targets

The ubiquitin-proteasome pathway plays a role in the degradation of the bulk of proteins in the cytoplasmic and nuclear compartments. In this pathway proteins are targeted for degradation by covalent ligation with ubiquitin, a reaction that requires ATP. Following the binding of the first ubiquitin molecule with the epsilon-amino group of a lysine residue of the substrate protein, a polyubiquitin chain is usually formed, in which the C-terminus of each ubiquitin unit is linked to a specific Lys residue of the previous ubiquitin. Central to this pathway is the 26S proteasome, a high molecular mass multifunctional protease which requires ATP for its catalytic activity. Substrates of the 26S proteasome are not only old or damaged proteins, but also short lived proteins functioning as regulatory factors in a large array of cellular processes, such as cell cycle progression, cell growth and gene expression, inflammatory response and immune surveillance. A number of inhibitors of the catalytic activity of proteasomes have been developed and successfully employed in the study of their functional and structural properties, as well as of their involvement in different cellular processes. Some of these molecules due to their toxicity are used only as experimental research tools; others instead are now in clinical trials for treatment of a variety of hematologic malignancies and solid tumors and of reperfusion injury occurring after cerebral ischemia and myocardial infarction. Furthermore, proteasome inhibitors are described to interfere with HIV maturation, budding and aggressiveness, and cytostatic drugs, as well as antiretroviral agents used in HAART, have been shown to behave in vitro and in cultured cell lines as inhibitors of proteasome proteolytic activity at therapeutic dosages.     

Proteasome inhibitors as anti-cancer agents

The ubiquitin (Ub)-proteasome pathway is the major nonlysosomal pathway of proteolysis in human cells and accounts for the degradation of most short-lived, misfolded or damaged proteins. This pathway is important in the regulation of a number of key biological regulatory mechanisms. Proteins are usually targeted for proteasome-mediated degradation by polyubiquitinylation, the covalent addition of multiple units of the 76 amino acid protein Ub, which are ligated to 1-amino groups of lysine residues in the substrate. Polyubiquitinylated proteins are degraded by the 26S proteasome, a large, ATP-dependent multicatalytic protease complex, which also regenerates monomeric Ub. The targets of this pathway include key regulators of cell proliferation and cell death. An alternative form of the proteasome, termed the immunoproteasome, also has important functions in the generation of peptides for presentation by MHC class I molecules. In recent years there has been a great deal of interest in the possibility that proteasome inhibitors, through elevation of the levels of proteasome targets, might prove useful as a novel class of anti-cancer drugs. Here we review the progress made to date in this area and highlight the potential advantages and weaknesses of this approach.     

蛋白酶体抑制剂的研究进展

作为体内蛋白质降解的途径之一,蛋白酶体具有非常重要的生理作用,并与多种疾病密切相关。抑制蛋白酶体的功能已经成为肿瘤治疗的叉一有前景的新途径,并受到越来越多的关注。本文对蛋白酶体的组成结构、病理生理作用和现有抑制剂进行归纳总结。     

TMC-86A, B and TMC-96, new proteasome inhibitors from Streptomyces sp. TC 1084 and Saccharothrix sp. TC 1094. I. Taxonomy, fermentation, isolation, and biological activities

TMC-86A, B and TMC-96, new 20S proteasome inhibitors with an epoxy-beta-aminoketone moiety, were isolated from the fermentation broth of Streptomyces sp. TC 1084 and Saccharothrix sp. TC 1094, respectively. TMC-86A, B and TMC-96 inhibited the chymotrypsin-like and peptidylglutamyl-peptide hydrolyzing activities of 20S proteasome with the following IC50 values: TMC-86A, 5.1 microM and 3.7microM; TMC-86B, 1.1 microM and 31 microM; TMC-96, 2.9 microM and 3.5 microM, respectively. TMC-86A, B and TMC-96 exhibited the weak inhibitory activity against the trypsin-like activity of 20S proteasome with IC50 values of 51 microM, 250 microM, and 36 microM, respectively. They did not inhibit m-calpain, cathepsin L, and trypsin at 100 microM, suggesting their high specificity for proteasome. Taxonomy of the producing strains is also described.     

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.