Modulation of proteasome subunit selectivity of syringolins.

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure-activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure-activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding ß5 and ß2 subunits.

Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2.

Rapid recovery of proteasome activity may contribute to intrinsic and acquired resistance to FDA-approved proteasome inhibitors. Previous studies have demonstrated that the expression of proteasome genes in cells treated with sub-lethal concentrations of proteasome inhibitors is upregulated by the transcription factor Nrf1 (NFE2L1), which is activated by a DDI2 protease. Here, we demonstrate that the recovery of proteasome activity is DDI2-independent and occurs before transcription of proteasomal genes is upregulated but requires protein translation. Thus, mammalian cells possess an additional DDI2 and transcription-independent pathway for the rapid recovery of proteasome activity after proteasome inhibition.

Histone deacetylase (HDAC) inhibitor specificity determinants are preserved in a class of dual HDAC/non-covalent proteasome inhibitors.

Many disease states require multiple drugs to inhibit multiple targets for their effective treatment/management, i.e. a drug cocktail regimen, or "polypharmacy". Polypharmacology, in contrast, is the development of single agents that can inhibit multiple targets. Each strategy is associated with advantages and disadvantages. Motivated by promising clinical trial data for the treatment of multiple myeloma with the combination of the HDAC6 inhibitor ricolinostat and the proteasome inhibitor bortezomib, we herein describe a focused family of dual HDAC/non-covalent proteasome inhibitors, and explore the impact of linker and zinc-binding group identities on HDAC1/6 isozyme selectivity. In general, previously reported specificity determinants of monovalent HDAC1/6 inhibitors were preserved in our dual HDAC/proteasome inhibitors.

Immunoproteasomal Processing of IsoLG-Adducted Proteins Is Essential for Hypertension.

BACKGROUND: Hypertension is characterized by CD8+ (cluster differentiation 8) T cell activation and infiltration into peripheral tissues. CD8+ T cell activation requires proteasomal processing of antigenic proteins. It has become clear that isoLG (isolevuglandin)-adduced peptides are antigenic in hypertension; however, IsoLGs inhibit the constitutive proteasome. We hypothesized that immunoproteasomal processing of isoLG-adducts is essential for CD8+ T cell activation and inflammation in hypertension. METHODS: IsoLG adduct processing was studied in murine dendritic cells (DCs), endothelial cells (ECs), and B8 fibroblasts. The role of the proteasome and the immunoproteasome in Ang II (angiotensin II)-induced hypertension was studied in C57BL/6 mice treated with bortezomib or the immunoproteasome inhibitor PR-957 and by studying mice lacking 3 critical immunoproteasome subunits (triple knockout mouse). We also examined hypertension in mice lacking the critical immunoproteasome subunit LMP7 (large multifunctional peptidase 7) specifically in either DCs or ECs. RESULTS: We found that oxidant stress increases the presence of isoLG adducts within MHC-I (class I major histocompatibility complex), and immunoproteasome overexpression augments this. Pharmacological or genetic inhibition of the immunoproteasome attenuated hypertension and tissue inflammation. Conditional deletion of LMP7 in either DCs or ECs attenuated hypertension and vascular inflammation. Finally, we defined the role of the innate immune receptors STING (stimulator of interferon genes) and TLR7/8 (toll-like receptor 7/8) as drivers of LMP7 expression in ECs. CONCLUSIONS: These studies define a previously unknown role of the immunoproteasome in DCs and ECs in CD8+ T cell activation. The immunoproteasome in DCs and ECs is critical for isoLG-adduct presentation to CD8+ T cells, and in the endothelium, this guides homing and infiltration of T cells to specific tissues.

Differential Interactions of the Proteasome Inhibitor PI31 with Constitutive and Immuno-20S Proteasomes.

PI31 (Proteasome Inhibitor of 31,000 Da) is a 20S proteasome binding protein originally identified as an in vitro inhibitor of 20S proteasome proteolytic activity. Recently reported cryo-electron microscopy structures of 20S-PI31 complexes have revealed that the natively disordered proline-rich C-terminus of PI31 enters the central chamber in the interior of the 20S proteasome and interacts directly with the proteasome's multiple catalytic threonine residues in a manner predicted to inhibit their enzymatic function while evading its own proteolysis. Higher eukaryotes express an alternative form of the 20S proteasome (termed "immuno-proteasome") that features genetically and functionally distinct catalytic subunits. The effect of PI31 on immuno-proteasome function is unknown. We examine the relative inhibitory effects of PI31 on purified constitutive (20Sc) and immuno-(20Si) 20S proteasomes in vitro and show that PI31 inhibits 20Si hydrolytic activity to a significantly lesser degree than that of 20Sc. Unlike 20Sc, 20Si hydrolyzes the carboxyl-terminus of PI31 and this effect contributes to the reduced inhibitory activity of PI31 toward 20Si. Conversely, loss of 20Sc inhibition by PI31 point mutants leads to PI31 degradation by 20Sc. These results demonstrate unexpected differential interactions of PI31 with 20Sc and 20Si and document their functional consequences.

Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy.

Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor-erythroid derived-2-related factor-1 (Nrf1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here, we report that under similar circumstances, multiple components of the autophagy-lysosomal pathway (ALP) were transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells, where Nrf1 is known to be non-functional. Conversely, overexpression of Nrf1 induced ALP genes and endowed the cells with an increased capacity to clear aggresomes. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress.

Proteasome Inhibition Reprograms Chromatin Landscape in Breast Cancer.

The 26S proteasome is the major protein degradation machinery in cells. Cancer cells use the proteasome to modulate gene expression networks that promote tumor growth. Proteasome inhibitors have emerged as effective cancer therapeutics, but how they work mechanistically remains unclear. Here, using integrative genomic analysis, we discovered unexpected reprogramming of the chromatin landscape and RNA polymerase II (RNAPII) transcription initiation in breast cancer cells treated with the proteasome inhibitor MG132. The cells acquired dynamic changes in chromatin accessibility at specific genomic loci termed differentially open chromatin regions (DOCR). DOCRs with decreased accessibility were promoter proximal and exhibited unique chromatin architecture associated with divergent RNAPII transcription. Conversely, DOCRs with increased accessibility were primarily distal to transcription start sites and enriched in oncogenic superenhancers predominantly accessible in non-basal breast tumor subtypes. These findings describe the mechanisms by which the proteasome modulates the expression of gene networks intrinsic to breast cancer biology. SIGNIFICANCE: Our study provides a strong basis for understanding the mechanisms by which proteasome inhibitors exert anticancer effects. We find open chromatin regions that change during proteasome inhibition, are typically accessible in non-basal breast cancers.

Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

Brain-Permeable Immunoproteasome-Targeting Macrocyclic Peptide Epoxyketones for Alzheimer's Disease.

Previously, we demonstrated that linear peptide epoxyketones targeting the immunoproteasome (iP) could ameliorate cognitive deficits in mouse models of Alzheimer's disease (AD) independently of amyloid deposition. We also reported the first iP-targeting macrocyclic peptide epoxyketones, which exhibit improved metabolic stability compared with their linear counterparts. Here, we prepared additional macrocyclic peptide epoxyketones and compared them with existing macrocyclic iP inhibitors by assessing Caco2 cell-based permeability and microsomal stability, providing the four best macrocyclic iP inhibitors. We then evaluated the four compounds using the Ames test and the potency assays in BV2 cells, selecting compound 5 as our AD drug lead. When 5 was administered intravenously (40 mg/kg) or orally (150 mg/kg) into healthy BALB/c mice, we observed considerable iP inhibition in the mouse brain, indicating good blood-brain barrier permeability and target engagement. Combined results suggest that 5 is a promising AD drug lead that may need further investigation.

Research strategies of small molecules as chemotherapeutics to overcome multiple myeloma resistance.

Multiple myeloma (MM), a cancer of plasma cells, is the second most common hematological malignancy which is characterized by aberrant plasma cells infiltration in the bone marrow and complex heterogeneous cytogenetic abnormalities. Over the past two decades, novel treatment strategies such as proteasome inhibitors, immunomodulators, and monoclonal antibodies have significantly improved the relative survival rate of MM patients. However, the development of drug resistance results in the majority of MM patients suffering from relapse, limited treatment options and uncontrolled disease progression after relapse. There are urgent needs to develop and explore novel MM treatment strategies to overcome drug resistance and improve efficacy. Here, we review the recent small molecule therapeutic strategies for MM, and introduce potential new targets and corresponding modulators in detail. In addition, this paper also summarizes the progress of multi-target inhibitor therapy and protein degradation technology in the treatment of MM.

MUC20 regulated by extrachromosomal circular DNA attenuates proteasome inhibitor resistance of multiple myeloma by modulating cuproptosis.

BACKGROUND: Proteasome inhibitors (PIs) are one of the most important classes of drugs for the treatment of multiple myeloma (MM). However, almost all patients with MM develop PI resistance, resulting in therapeutic failure. Therefore, the mechanisms underlying PI resistance in MM require further investigation. METHODS: We used several MM cell lines to establish PI-resistant MM cell lines. We performed RNA microarray and EccDNA-seq in MM cell lines and collected human primary MM samples to explore gene profiles. We evaluated the effect of MUC20 on cuproptosis of PI-resistant MM cells using Co-immunoprecipitation (Co-IP), Seahorse bioenergetic profiling and in vivo assay. RESULTS: This study revealed that the downregulation of Mucin 20 (MUC20) could predict PI sensitivity and outcomes in MM patients. Besides, MUC20 attenuated PI resistance in MM cells by inducing cuproptosis via the inhibition of cyclin-dependent kinase inhibitor 2 A expression (CDKN2A), which was achieved by hindering MET proto-oncogene, receptor tyrosine kinase (MET) activation. Moreover, MUC20 suppressed MET activation by repressing insulin-like growth factor receptor-1 (IGF-1R) lactylation in PI-resistant MM cells. This study is the first to perform extrachromosomal circular DNA (eccDNA) sequencing for MM, and it revealed that eccDNA induced PI resistance by amplifying kinesin family member 3 C (KIF3C) to reduce MUC20 expression in MM. CONCLUSION: Our findings indicated that MUC20 regulated by eccDNA alleviates PI resistance of MM by modulating cuproptosis, which would provide novel strategies for the treatment of PI-resistant MM.

GZ17-6.02 interacts with proteasome inhibitors to kill multiple myeloma cells.

GZ17-6.02, a synthetically manufactured compound containing isovanillin, harmine and curcumin, has undergone phase I evaluation in patients with solid tumors (NCT03775525) with a recommended phase 2 dose (RP2D) of 375 mg PO BID. GZ17-6.02 was more efficacious as a single agent at killing multiple myeloma cells than had previously been observed in solid tumor cell types. GZ17-6.02 interacted with proteasome inhibitors in a greater than additive fashion to kill myeloma cells and alone it killed inhibitor-resistant cells to a similar extent. The drug combination of GZ17-6.02 and bortezomib activated ATM, the AMPK and PERK and inactivated ULK1, mTORC1, eIF2α, NFκB and the Hippo pathway. The combination increased ATG13 S318 phosphorylation and the expression of Beclin1, ATG5, BAK and BIM, and reduced the levels of BCL-XL and MCL1. GZ17-6.02 interacted with bortezomib to enhance autophagosome formation and autophagic flux, and knock down of ATM, AMPKα, ULK1, Beclin1 or ATG5 significantly reduced both autophagy and tumor cell killing. Knock down of BAK and BIM significantly reduced tumor cell killing. The expression of HDACs1/2/3 was significantly reduced beyond that previously observed in solid tumor cells and required autophagy. This was associated with increased acetylation and methylation of histone H3. Combined knock down of HDACs1/2/3 caused activation of ATM and the AMPK and caused inactivation of ULK1, mTORC1, NFκB and the Hippo pathway. HDAC knock down also enhanced ATG13 phosphorylation, increased BAK levels and reduced those of BCL-XL. Collectively, our present studies support performing additional in vivo studies with multiple myeloma cells.

Cisplatin Disrupts Proteasome Bounce-Back Effect through Suppressing ZEB1/Nfe2l1 in Cholangiocarcinoma.

BACKGROUND: Bortezomib (BTZ) is a powerful proteasome inhibitor that has been approved for the treatment of haematologic malignancies. Its effectiveness has been assessed against different types of solid tumours. BTZ is ineffective in most solid tumours because of drug resistance, including cholangiocarcinoma, which is associated with a proteasome bounce-back effect. However, the mechanism through which proteasome inhibitors induce the proteasome bounce-back effect remains largely unknown. METHODS: Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry. RESULTS: Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells. CONCLUSIONS: Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.

TRAIL-induced apoptosis and proteasomal activity - Mechanisms, signalling and interplay.

Programmed cell death, in particular apoptosis, is essential during development and tissue homeostasis, and also is the primary strategy to induce cancer cell death by cytotoxic therapies. Precision therapeutics targeting TRAIL death receptors are being evaluated as novel anti-cancer agents, while in parallel highly specific proteasome inhibitors have gained approval as drugs. TRAIL-dependent signalling and proteasomal control of cellular proteostasis are intricate processes, and their interplay can be exploited to enhance therapeutic killing of cancer cells in combination therapies. This review provides detailed insights into the complex signalling of TRAIL-induced pathways and the activities of the proteasome. It explores their core mechanisms of action, pharmaceutical druggability, and describes how their interplay can be strategically leveraged to enhance cell death responses in cancer cells. Offering this comprehensive and timely overview will allow to navigate the complexity of the processes governing cell death mechanisms in TRAIL- and proteasome inhibitor-based treatment conditions.

Targeting myeloma metabolism: How abnormal metabolism contributes to multiple myeloma progression and resistance to proteasome inhibitors.

Multiple myeloma is a hematological malignancy that has evolved from antibody-secreting B lymphocytes. Like other types of cancers, myeloma cells have acquired functional capabilities which are referred to as "Hallmarks of Cancer", and one of their most important features is the metabolic disorders. Due to the high secretory load of the MM cells, the first-line medicine proteasome inhibitors have found their pronounced effects in MM cells for blocking the degradation of misfolded proteins, leading to their accumulation in the ER and overwhelming ER stress. Moreover, proteasome inhibitors have been reported to be effective in myeloma by targeting glucose, lipid, amino acid metabolism of MM cells. In this review, we have described the abnormal metabolism of the three major nutrients, such as glucose, lipid and amino acids, which participate in the cellular functions. We have described their roles in myeloma progression, how they could be exploited for therapeutic purposes, and current therapeutic strategies targeting these metabolites, hoping to uncover potential novel therapeutic targets and promote the development of future therapeutic approaches.

Interplay between proteasome inhibitors and NF-κB pathway in leukemia and lymphoma: a comprehensive review on challenges ahead of proteasome inhibitors.

The current scientific literature has extensively explored the potential role of proteasome inhibitors (PIs) in the NF-κB pathway of leukemia and lymphoma. The ubiquitin-proteasome system (UPS) is a critical component in regulating protein degradation in eukaryotic cells. PIs, such as BTZ, are used to target the 26S proteasome in hematologic malignancies, resulting in the prevention of the degradation of tumor suppressor proteins, the activation of intrinsic mitochondrial-dependent cell death, and the inhibition of the NF-κB signaling pathway. NF-κB is a transcription factor that plays a critical role in the regulation of apoptosis, cell proliferation, differentiation, inflammation, angiogenesis, and tumor migration. Despite the successful use of PIs in various hematologic malignancies, there are limitations such as resistant to these inhibitors. Some reports suggest that PIs can induce NF-κB activation, which increases the survival of malignant cells. This article discusses the various aspects of PIs' effects on the NF-κB pathway and their limitations. Video Abstract.

Protocol for analysis of intracellular conversion of artezomib molecules into new proteasome inhibitors in Plasmodium falciparum parasites.

Artezomibs (ATZs), dual-pharmacophore molecules comprising of artemisinin and a parasite proteasome inhibitor, hijack parasite ubiquitin proteasome system to transform into new proteasome inhibitors following the activation of artemisinin by heme.1 Here, we present a protocol for using a fluorescent activity-based broad-spectrum proteasome inhibitor probe to study intracellular conversion of ATZ molecules into new proteasome inhibitors in malaria parasites. We describe steps for drug treatment and washout, parasite lysis, proteasome labeling, and visualization. For complete details on the use and execution of this protocol, please refer to Zhan et al.1.

Autophagy and oxidative stress modulation mediate Bortezomib resistance in prostate cancer.

Proteasome inhibitors such as Bortezomib represent an established type of targeted treatment for several types of hematological malignancies, including multiple myeloma, Waldenstrom's macroglobulinemia, and mantle cell lymphoma, based on the cancer cell's susceptibility to impairment of the proteasome-ubiquitin system. However, a major problem limiting their efficacy is the emergence of resistance. Their application to solid tumors is currently being studied, while simultaneously, a wide spectrum of hematological cancers, such as Myelodysplastic Syndromes show minimal or no response to Bortezomib treatment. In this study, we utilize the prostate cancer cell line DU-145 to establish a model of Bortezomib resistance, studying the underlying mechanisms. Evaluating the resulting resistant cell line, we observed restoration of proteasome chymotrypsin-like activity, regardless of drug presence, an induction of pro-survival pathways, and the substitution of the Ubiquitin-Proteasome System role in proteostasis by induction of autophagy. Finally, an estimation of the oxidative condition of the cells indicated that the resistant clones reduce the generation of reactive oxygen species induced by Bortezomib to levels even lower than those induced in non-resistant cells. Our findings highlight the role of autophagy and oxidative stress regulation in Bortezomib resistance and elucidate key proteins of signaling pathways as potential pharmaceutical targets, which could increase the efficiency of proteasome-targeting therapies, thus expanding the group of molecular targets for neoplastic disorders.

The effect of immunomodulatory drugs on bone metabolism of patients with multiple myeloma.

INTRODUCTION: Immunomodulatory drugs (IMiDs) are widely used in the management of newly diagnosed and relapsed/refractory multiple myeloma patients. These agents show their potential effect on myeloma bone disease (MBD), including inhibition of osteoclasts activity and effects on osteoblasts differentiation. It is unclear whether these effects are direct, which may have an impact on bone formation markers when combined with proteasome inhibitors. AREAS COVERED: This review summarizes the available evidence on the role of IMiDs in microenvironment regulation and their potential effects on bone metabolism. The literature search methodology consisted of searching PubMed for basic and clinical trials using medical subject terms. Included articles were screened and evaluated by the coauthors of this review. EXPERT OPINION: As a therapeutic option, IMiDs directly affect preosteoblast/osteoclast differentiation. The combination of proteasome inhibitors may counteract the short-term up-regulation of osteogenic activity markers, and therefore intravenous zoledronic acid is recommended, however, obtaining a more significant myeloma response will have a long-term positive impact on myeloma bone disease.

In vivo activity of the second-generation proteasome inhibitor ixazomib against pediatric T-cell acute lymphoblastic leukemia xenografts.

The overall survival rate of patients with T-cell acute lymphoblastic leukemia (T-ALL) is now 90%, although patients with relapsed T-ALL face poor prognosis. The ubiquitin-proteasome system maintains normal protein homeostasis, and aberrations in this pathway are associated with T-ALL. Here we demonstrate the in vitro and in vivo activity of ixazomib, a second-generation orally available, reversible, and selective proteasome inhibitor against pediatric T-ALL cell lines and patient-derived xenografts (PDXs) grown orthotopically in immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJAusb (NSG) mice. Ixazomib was highly potent in vitro, with half-maximal inhibitory concentration (IC50) values in the low nanomolar range. As a monotherapy, ixazomib significantly extended mouse event-free survival of five out of eight T-ALL PDXs in vivo.