Proteasomal Enzyme Research Articles

Discovery of ester-linked ubiquitylation of PARP10 mono-ADP-ribosylation in cells: a dual post-translational modification on Glu/Asp side chains.

The prevailing view on post-translational modifications (PTMs) is that amino acid side chains in proteins are modified with a single PTM at any given time. However, a growing body of work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation, where specialized E3 ligases ubiquitylate targets for proteasomal degradation in an ADP-ribosylation-dependent manner. More recently, the DELTEX family of E3 ligases was reported to catalyze ubiquitylation of the 3'- hydroxy group of the adenine-proximal ribose of free NAD + and ADP-ribose in vitro , generating a non-canonical ubiquitin ester-linked species. In this report, we show, for the first time, that this dual PTM occurs in cells on mono-ADP-ribosylated (MARylated) PARP10 on Glu/Asp sites to form a MAR ubiquitin ester (MARUbe). We term this process m ono- A DP-ribosyl ub iquit ylation or MARUbylation. Using chemical and enzymatic treatments, including a newly characterized bacterial deubiquitinase with esterase-specific activity, we discovered that PARP10 MARUbylation is extended with K11-linked polyubiquitin chains. Finally, mechanistic studies using proteasomal and ubiquitin-activating enzyme inhibitors demonstrated that PARP10 MARUbylation leads to its proteasomal degradation, providing a functional role for this new PTM in regulating protein turnover.

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

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. 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. 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. 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.

A ubiquitin-proteasome system-related signature to predict prognosis, immune infiltration, and therapy efficacy for breast cancer.

The ubiquitin-proteasome system (UPS) is an essential regulatory system for maintaining homeostasis, and its dysfunction may cause various diseases. The activity of proteasome and ubiquitin-conjugating enzymes has been found to be greatly increased in breast cancer (BC), indicating that the heterogeneity of UPS may be related to the progression of BC. Gene data was obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases and performed in multiple algorithms to construct a UPS-related signature for BC. Patients in the UPS low-risk group had greater overall and recurrence-free survival probability than those in the UPS high-risk group. This signature was closely associated with functional enrichment. Some high metabolism-related pathways were more active in the UPS high-risk group. The UPS low-risk group had more abundant anti-tumor immune cells, while in the UPS high-risk group, immunosuppressive cells were dominant. More importantly, we found that the UPS low-risk group was more sensitive to immunotherapy, while the UPS high-risk group responded better to radiotherapy. Drug sensitivity analysis identified more effective chemotherapy drugs in different UPS-related risk groups. This UPS-related signature may serve as a novel biomarker and independent prognostic factor for BC. It can effectively predict prognosis, immune infiltration, and therapy efficacy, providing new strategies for individualized treatment.

Growth retardation and suppression of ubiquitin-dependent catabolic processes in the brackish water clam Corbicula japonica in response to salinity changes and bioaccumulation of toxic heavy metals

The brackish water clam (Corbicula japonica) is constantly exposed to stressful salinity gradients and high levels of heavy metals in the freshwater-saltwater interface of estuary environments, which are introduced from upstream regions and land. To identify the key molecular pathways involved in the response to salinity changes and heavy metal bioaccumulation, we obtained the transcriptomes of C. japonica inhabiting different salinities and heavy metal distributions in Gwangyang Bay (Korea) using RNA sequencing. Among a total of 404,486 assembled unigenes, 5534 differentially expressed genes were identified in C. japonica inhabiting different conditions, 1549 of which were significantly upregulated and 1355 were significantly downregulated. Correlation analyses revealed distinct gene expression patterns between the low and high conditions of salinity and heavy metal bioaccumulation. Functional annotation revealed significant downregulation of genes involved in “ubiquitin-dependent protein catabolic process,” “tricarboxylic acid cycle,” and “intracellular protein transport” in C. japonica from the high condition compared to the low condition. Transcription and translation pathways were significantly enriched in the high condition. Additionally, upon comparison of the low and high conditions by qRT-PCR and proteasome enzyme activity analyses, our findings demonstrated that environmental stress could suppress the ubiquitin-proteasome complex (UPC). Additionally, transcriptomic changes under high salinity stress conditions may be related to an increase in cellular protection by defense enzymes, which leads to more energy being required and a disruption of energy homeostasis. Ultimately, this could cause growth retardation in the clam C. japonica. In summary, this study provides the first evidence of UPC suppression induced by a combination of high salinity and heavy metal bioaccumulation stress in C. japonica, which could compromise the survival and growth of estuarine bivalves.

Ubiquitin-Conjugating Enzymes Ubc1 and Ubc4 Mediate the Turnover of Hap4, a Master Regulator of Mitochondrial Biogenesis in Saccharomyces cerevisiae.

Mitochondrial biogenesis is tightly regulated in response to extracellular and intracellular signals, thereby adapting yeast cells to changes in their environment. The Hap2/3/4/5 complex is a master transcriptional regulator of mitochondrial biogenesis in yeast. Hap4 is the regulatory subunit of the complex and exhibits increased expression when the Hap2/3/4/5 complex is activated. In cells grown under glucose derepression conditions, both the HAP4 transcript level and Hap4 protein level are increased. As part of an inter-organellar signaling mechanism coordinating gene expression between the mitochondrial and nuclear genomes, the activity of the Hap2/3/4/5 complex is reduced in respiratory-deficient cells, such as ρ0 cells lacking mitochondrial DNA, as a result of reduced Hap4 protein levels. However, the underlying mechanism is unclear. Here, we show that reduced HAP4 expression in ρ0 cells is mediated through both transcriptional and post-transcriptional mechanisms. We show that loss of mitochondrial DNA increases the turnover of Hap4, which requires the 26S proteasome and ubiquitin-conjugating enzymes Ubc1 and Ubc4. Stabilization of Hap4 in the ubc1 ubc4 double mutant leads to increased expression of Hap2/3/4/5-target genes. Our results indicate that mitochondrial biogenesis in yeast is regulated by the functional state of mitochondria partly through ubiquitin/proteasome-dependent turnover of Hap4.

Phase I Study of a Novel Oral Proteasome Inhibitor TQB3602 in Relapsed/Refractory Multiple Myeloma

Background: TQB3602 is a novel oral proteasome inhibitor with an acridine ring in addition to the main structure of ixazomib, which maintains better metabolic stability and higher activity and selectivity in preclinical models of multiple myeloma. TQB3602 consists of the L-malate ester with active boronic acid compound WXFL10410333 selectively targeting 20S proteasome enzyme. This is the first-in-human study to evaluate the safety, tolerability and pharmacokinetics of TQB3602 and assess the efficacy of TQB3602 combined with immunomodulators/dexamethasone regimen in relapsed/refractory multiple myeloma (RRMM). Study Design and Methods: This study is a multi-center open-label Phase I study in RRMM, consisting of the dose escalation phase and dose expansion phase. The inclusion criteria mainly include: 1) 18 to 75 years old male or female, 2) ECOG score of 0 to 2, 3) Life expectancy ≥12 weeks, 4) RRMM had received at least two treatment regimens (including lenalidomide or thalidomide, bortezomib, and glucocorticoid). The exclusion criteria mainly include: 1) grade ≥ 2 peripheral nerve diseases, 2) grade > 1 diarrhea during the screening period, 3) ixazomib has been administered less than five half-lives before the first dose of TQB3602, 4) previous allogeneic hematopoietic stem cell transplantation. TQB3602 was administered alone in 3+3 dose escalation (at a starting dose of 0.5mg on days 1, 8 and 15 in a 28-day cycle) until toxicity or disease progression in the dose escalation phase. According to prior lines of therapy, patients with RRMM were divided into two dose expansion cohorts, including TQB3602/lenalidomide/dexamethasone and TQB306/pomalidomide/dexamethasone (TQB3602 6mg on days 1, 8, 15, lenalidomide 25mg daily or pomalidomide 4mg daily on days 1 to 21, dexamethasone 20mg on days 1-2, 8-9, 15-16, 22-23 in a 28-day cycle). Results: The study enrolled 56 patients with RRMM including 25 patients in the dose escalation and 31 patients in the dose expansion between May 28, 2020 and July 20, 2022. The demographics and baseline characteristics were shown in table 1. Patients received single-agent TQB 3602 0.5mg to 7 mg on days 1, 8 and 15 of a 28-day cycle and two dose-limiting toxicities occurred at 7 mg, including diarrhea and grade 2 peripheral neuropathy. The maximum tolerated dose of TQB3602 was determined as 6.0mg. The terminal half-life was 23 to 152 hours after multiple dosing and the mean half-life was approximately 82 hours for 6mg TQB3602. The dose expansion cohorts recruited 29 RRMM patients receiving TQB3602/pomalidomide/dexamethasone regimen and two RRMM patients receiving TQB3602/lenalidomide/dexamethasone regimen. Among 14 response-evaluable patients in the dose expansion cohorts, eight RRMM (57%) achieved partial response or better (figure 1). The most common adverse events included thrombocytopenia, neutropenia, anemia, diarrhea, hypokalemia, fatigue and peripheral neuropathy. Adverse events of grade ≥3 included thrombocytopenia, neutropenia, anemia, elevated transaminase, diarrhea and pulmonary infection. Conclusions: TQB3602 is generally well tolerated. The combination therapy has shown preliminary efficacy in RRMM, and further clinical trials are in progress. (NCT04275583) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparative proteomic analysis reveals differential protein expression of Hypsizygus marmoreus in response to different light qualities

Light is important environmental stress that influences the growth, development, and metabolism of Hypsizygus marmoreus (white var.). However, the molecular basis of the light effect on H. marmoreus remains unclear. In this study, a label-free comparative proteomic analysis was applied to investigate the global protein expression profile of H. marmoreus mycelia growing under white, red, green, and blue light qualities and darkness (control). Among 3149 identified proteins in H. marmoreus, 2288 were found to be expressed in all tested conditions. Data of Each light quality was compared with darkness for further analysis, numerous differentially expressed proteins (DEPs) were identified and the white light group showed the most. All the up-regulated and down-regulated DEPs were annotated and analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The KEGG enrichment analysis revealed that light stress was associated with primary metabolism, glycolysis/gluconeogenesis, MAPK, proteasome, and carbohydrate-active enzyme pathways. This study advances valuable insights into the molecular mechanisms underlying the role of different light qualities in mushroom growth and development.

292 (PB072) - Development of selective MCL-1 heterobifunctional degraders

Background: MCL1 is a member of the Bcl-2 family that plays a key role in cellular homeostasis through the regulation of apoptosis as well as other less characterized functions. The growing recognition of MCL-1 role in cancer cell survival and its association with the development of anticancer drug resistance makes it an attractive target for cancer therapy. Several inhibitors have been developed during the past decade and some of them have entered clinical trials, but no drugs have been approved for clinical use so far. Targeted protein degradation using heterobifunctional degraders has emerged as a novel therapeutic modality in drug discovery. This technology has many potential advantages over traditional inhibitors, e.g. reduced side effects and drug resistance, extended duration of action, elevated selectivity, and the opportunity to target “undruggable” proteins. In this study, we report the development of the first-in-class MCL-1 bifunctional degraders with in vivo PD effect. Material and Methods: Compounds’ affinity to CRBN (Cereblon), an E3 ligase, and to MCL-1 was determined by FP and SPR, respectively. AlphaLISA and HTRF were utilized to confirm ternary complex formation. Cell viability was assessed by CellTiter-Glo Assay. Apoptosis induction and changes in mitochondrial membrane potential were assessed by flow cytometry using Annexin/PI and TMRE staining, respectively. Targeted protein degradation in cells treated with compounds in the absence or presence of apoptosis inhibitor was assessed by WB. The mechanism of action of the compounds was confirmed in cancer cells pre-treated with proteasome or NEDD8-Activating Enzyme inhibitors, or CRBN-targeting siRNA, and then treated with the compounds. MCL-1 abundance and expression of apoptosis markers were confirmed by WB. PK studies were conducted in CD1 mice and Sprague Dawley rats, and PD studies in CB-17 SCID mice xenografted with MV4–11 cells. Results: Synthesized compounds bind CRBN and MCL-1 with high affinity, form a ternary complex in vitro and induce the degradation of MCL-1 in cells, which is caspase- and proteasome-dependent and leads to apoptosis induction. These series of compounds showed a nM range cytotoxic activity in haematological cancer cell lines with the best compounds having an IC50 <100 nM. Moreover, MCL-1 degradation in vivo can be achieved by IP administration leading to the initiation of the programmed cell death. Conclusions: Our compounds have a broad cytotoxic activity toward cultured human cancer cells and a promising in vivo pharmacokinetic profile. Our data indicate that targeting MCL-1 by employing bifunctional degraders represents a potentially new and effective strategy for cancer treatment. No conflict of interest.

α-mangostin derivative 4e as a PDE4 inhibitor promote proteasomal degradation of alpha-synuclein in Parkinson's disease models through PKA activation

BackgroundParkinson's disease (PD) is a multi-factorial neurodegenerative disease affecting motor function of patients. The hall markers of PD are dopaminergic neuron loss in the midbrain and the presence of intra-neuronal inclusion bodies mainly composed of aggregation-prone protein alpha-synuclein (α-syn). Ubiquitin-proteasome system (UPS) is a multi-step reaction process responsible for more than 80% intracellular protein degradation. Impairment of UPS function has been observed in the brain tissue of PD patients. PDE4 inhibitors have been shown to activate cAMP-PKA pathway and promote UPS activity in Alzheimer's disease model. α-mangostin is a natural xanthonoid with broad biological activities, such as antioxidant, antimicrobial and antitumour activities. Structure-based optimizations based on α-mangostin produced a potent PDE4 inhibitor, 4e. Herein, we studied whether 4e could promote proteasomal degradation of α-syn in Parkinson's disease models through PKA activation. MethodscAMP Assay was conducted to quantify cAMP levels in samples. Model UPS substrates (Ub-G76V-GFP and Ub-R-GFP) were used to monitor UPS-dependent activity. Proteasome activity was investigated by short peptide substrate, Suc-LLVY-AMC, cleavage of which by the proteasome increases fluorescence sensitivity. Tet-on WT, A30P, and A53T α-syn-inducible PC12 cells and primary mouse cortical neurons from A53T transgenic mice were used to evaluate the effect of 4e against α-syn in vitro. Heterozygous A53T transgenic mice were employed to assess the effect of 4e on the clearance of α-syn in vivo, and further validations were applied by western blotting and immunohistochemistry. ResultsTaken together, α-mangostin derivative 4e, a PDE4 inhibitor, efficiently activated the cAMP/PKA pathway in neuronal cells, and promoted UPS activity as evidenced by enhanced degradation of UPS substrate Ub-G76V-GFP and Ub-R-GFP, as well as elevated proteasomal enzyme activity. Interestingly, 4e dramatically accelerated degradation of inducibly-expressed WT and mutant α-syn in PC12 cells, in a UPS dependent manner. Besides, 4e consistently activated PKA in primary neuron and A53T mice brain, restored UPS inhibition and alleviated α-syn accumulation in the A53T mice brain. Conclusions4e is a natural compound derived highly potent PDE4 inhibitor. We revealed its potential effect in promoting UPS activity to degrade pathogenic proteins associated with PD.

TMOD-10. THE ROLE OF IDH1 R132H MUTATION IN GLIOMA – AN INVESTIGATION BY GENOME-EDITING IN HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract BACKGROUND Hot-spot mutations in the Isocitrate dehydrogenase 1 (IDH1) cause a new catalytic function resulting in the production of 2-HG, a hallmark in the development of low-grade glioma. The tumorigenic mechanism of this mutation as well as the cell of origin are not known and there is a lack of suitable disease models. Thus, we aim to create a model mimicking glioma development by introducing the IDH1 R132H into human induced pluripotent stem cells (hiPSC) and investigate the influence on stem cell properties and cell differentiation in neuronal progenitor cells. MATERIAL AND METHODS We use CRISPR/Cas9 based genome editing to induce the IDH1 R132H mutation into healthy-control-derived hiPSCs. Successful introduction of the mutation was confirmed on DNA, RNA and protein level. The hiPSCs are then differentiated into cerebral organoids and characterized using transcriptome sequencing and methylation arrays. RESULTS We successfully introduced the IDH1 R132H mutation into hiPSCs and confirmed expression of the mutated protein by Western Blot. Metabolite measurement using liquid chromatography tandem mass spectrometry (LC-MS/MS) showed a forty times increased concentration of 2-HG in IDH-mutated compared to the wildtype hiPSCs, proving that the mutated enzyme is functional. To investigate effects of IDH1 R132H on cell differentiation, we generated cerebral organoids from our iPSC-models. The IDH1 R132H mutation did not inhibit cell differentiation or maturation of cerebral organoids but led to a downregulation of splicosome, proteasome and DNA repair enzymes as well as an upregulation of ECM components. CONCLUSION AND OUTLOOK hiPSCs with R132H mutation pose a promising model for investigations on early glioma development. We are currently step-wise including TP53 and ATRX loss of function mutations in our hiPSC models to recapitulating tumor development in vivo.

Neurotoxicity of a pyrethroid pesticide deltamethrin is associated with the imbalance in proteolytic systems caused by mitophagy activation and proteasome inhibition

Pyrethroids are one of the most commonly used classes of synthetic pesticides in the world. Recent laboratory and epidemiological evidence suggested that pyrethroids have potential adverse effects in the mammalian brain; however, the underlying mechanisms of the neurotoxic effects of pyrethroids have not been fully elucidated. In the present study, we investigated the mechanisms of effects of a type II pyrethroid deltamethrin (DM) in a neuronal cell model focusing on the proteolytic function, including autophagy and the ubiquitin-proteasome system. We confirmed that a micromolar concentration of DM dose-dependently decreased the cell viability and induced apoptotic cell death. Our results showed that DM enhanced autophagy in association with an accumulation of autophagosomes and increase in the levels of autophagy markers LC3-II/LC3-I ratio and p62 which were much elevated in the presence of lysosomal inhibitors bafilomycin A1 and chloroquine. We also found that DM caused a dysfunction of mitochondria with a decrease of mitochondrial membrane potential and mitochondrial DNA copy number as well as colocalization with autophagosomes. Moreover, a decrease in the activities of three major proteasomal enzymes and an accumulation of ubiquitinated proteins were observed by the exposure to DM. Transcriptome analysis revealed that up-regulated genes supported the activation of autophagy with induction of cellular stress responses including oxidative stress and endoplasmic reticulum stress, while down-regulated genes related to the cell cycle and DNA replication. These findings provide novel insights into the neurotoxicity of DM which underlie the imbalance in proteolytic function caused by mitophagy activation and proteasome inhibition.

Gene Expression Profiling of Lymph Node Sub-Capsular Sinus Macrophages in Cancer.

Lymph nodes are key lymphoid organs collecting lymph fluid and migratory cells from the tissue area they survey. When cancerous cells arise within a tissue, the sentinel lymph node is the first immunological organ to mount an immune response. Sub-capsular sinus macrophages (SSMs) are specialized macrophages residing in the lymph nodes that play important roles as gatekeepers against particulate antigenic material. In the context of cancer, SSMs capture tumor-derived extracellular vesicles (tEVs), a form of particulate antigen released in high amounts by tumor cells. We and others have recently demonstrated that SSMs possess anti-tumor activity because in their absence tumors progress faster. A comprehensive profiling of SSMs represents an important first step to identify the cellular and molecular mechanisms responsible for SSM anti-tumor activity. Unfortunately, the isolation of SSMs for molecular analyses is very challenging. Here, we combined an optimized dissociation protocol, careful marker selection and stringent gating strategies to highly purify SSMs. We provide evidence of decreased T and B cell contamination, which allowed us to reveal the gene expression profile of this elusive macrophage subset. Squamous cell carcinomas induced an increase in the expression of Fc receptors, lysosomal and proteasomal enzymes in SSMs. Imaging of mouse and patient lymph nodes confirmed the presence of the top differentially expressed genes. These results suggest that SSMs respond to tumor formation by upregulating the machinery necessary for presentation of tumor particulate antigens to B cells.

Autophagy regulates long-term cross-presentation by murine dendritic cells.

Autophagy has been reported to be involved in supporting antigen cross‐presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endolysosomal compartments and thereby sustain MHCI antigen cross‐presentation to CD8+ T cells. In the current study, we investigated the role of autophagy in long‐term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5–/–DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross‐presentation. This augmented antigen cross‐presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long‐term MHCI cross‐presentation.

An antibody-based amperometric biosensor for 20S proteasome activity and inhibitor screening.

The 20S proteasome enzyme complex is involved in the proteolytic degradation of misfolded and oxidatively damaged proteins and is a focus of medical research for the development of compounds with pharmaceutical properties, which are active in cancer cells and/or neurodegenerative diseases. The present study aims to develop a biosensor for investigating the 20S proteasome activity and inhibition by means of electrochemical methods. The 20S proteasome is best immobilized at the electrode surface through bio-affinity interactions with antibodies that target different subunits on the 20S proteasome, enabling the investigation of the effect of an enzyme's orientation on biosensor response. The enzymatic activity is analyzed by fixed potential amperometry with the highest sensitivity of 24 μA cm-2 mM-1 and a LOD of 0.4 μM. The detection principle involves the oxidation of an electroactive probe that is released from the enzyme's substrates upon proteolysis. The most sensitive biosensor is then used to study the multicatalytic activity of the 20S proteasome, i.e. the caspase-, trypsin- and chymotrypsin-like activity, by analyzing the biosensor's sensitivity towards different substrates. The behavior of the immobilized 20S proteasome is investigated as a function of substrate concentration. The kinetic parameters are derived and compared with those obtained when the enzyme was free in solution, with K0.5 values being one to two orders of magnitude lower in the present case. Two 20S inhibitors, epoxomicin and bortezomib, are investigated by analyzing their influence on the 20S biosensor response. The proposed analytical method for proteasome activity and inhibitor screening has the main advantage of being cost-effective compared to the ones typically employed.

Preclinical evaluation of TQB3602, a small molecule proteasome inhibitor clinical candidate.

e20546 Background: Proteasome inhibitors, selectively inhibit the activity of proteasome in ubiquitin proteasome system, together with immunomodulatory drugs formed the basis of many treatment regimens for multiple myeloma in first-line therapy as well as for the treatment of relapsed disease. Bortezomib (Velcade), the first approved therapeutic proteasome inhibitor with a unique boronic acid as warhead, limited by its requirement for parenteral administration (intravenous or subcutaneous). The 2nd generation boronic acid – orally bioavailable Ixazomib (Ninlaro) was approved in 2015 by FDA greatly improved patient’s compliance. However, its adverse effect remained an issue in clinical application. The purpose of this study is to investigate the in vitro and in vivo antitumor activity of TQB3602, a novel orally bioavailable small molecule proteasome inhibitor, in preclinical models of multiple myeloma. Methods: The enzyme inhibition and anti-proliferative activity of TQB3602 was evaluated in 20S proteasome and MM.1S cell line. Oral administration of TQB3602 at 3 doses was used to evaluate the in vivo anti-tumor activity in MM.1S multiple myeloma xenograft (CDX) models. Results: TQB3602 displayed potent kinase inhibiting activity for 20S proteasome enzyme with IC50 15.3 nM. TQB3602 inhibited cell proliferation in MM.1S multiple myeloma cell line with IC50 10.1 nM. In the MM.1S multiple myeloma CB-17 CDX model, TQB3602 showed antitumor efficacy with TGI &gt; 100% @7 mpk, BIW (tumor can’t be measured from day 17). Conclusions: We have identified a novel potent proteasome inhibitor TQB3602. Preclinical studies showed antitumor efficacy of TQB3602 in MM.1S multiple myeloma model. TQB3602 represents a promising clinical candidate for treating multiple myeloma and the First-in-human trial is expected in the middle of 2020.

Molecular Approaches to Treating Pediatric Leukemias.

Over the past decades, striking progress has been made in the treatment of pediatric leukemia, approaching 90% overall survival in children with acute lymphoblastic leukemia (ALL) and 75% in children with acute myeloid leukemia (AML). This has mainly been achieved through multiagent chemotherapy including CNS prophylaxis and risk-adapted therapy within collaborative clinical trials. However, prognosis in children with refractory or relapsed leukemia remains poor and has not significantly improved despite great efforts. Hence, more effective and less toxic therapies are urgently needed. Our understanding of disease biology, molecular drivers, drug resistance and, thus, the possibility to identify children at high-risk for treatment failure has significantly improved in recent years. Moreover, several new drugs targeting key molecular pathways involved in leukemia development, cell growth, and proliferation have been developed and approved. These striking achievements are linked to the great hope to further improve survival in children with refractory and relapsed leukemia. This review gives an overview on current molecularly targeted therapies in children with leukemia, including kinase, and proteasome inhibitors, epigenetic and enzyme targeting, as well as apoptosis regulators among others.

Electrochemical assay for 20S proteasome activity and inhibition with anti-cancer drugs

The majority of eukaryotic regulated protein turnover is performed by the proteasome, a multi-catalytic enzyme. Due to the fact that proteasome enzyme abnormal functioning was observed in different malignant cells, the proteasome is becoming a target for medical treatment. In order to evaluate the mechanisms of action of pharmaceutical compounds on proteasome enzyme inhibition, detecting and characterizing its activity is essential. An electrochemical assay that allows the monitoring of the chymotrypsin-like activity and inhibition of the 20S proteasome enzyme, based on the electrochemical detection of an electroactive compound released upon proteolysis of an adequate chymotrypsin-substrate is described. By employing differential pulse voltammetric measurement, the activity of the 20S proteasome enzyme was investigated for different incubation times of 20S with oligopeptide substrate as well as for different concentrations of substrate. Enzyme kinetic parameters were determined by voltammetry and the electrochemical assay compared with fluorescence spectroscopy. Electrochemical quartz crystal microbalance and atomic force microscopy were also used to investigate substrate interaction with the 20S proteasome and their adsorption at the electrode surface. Finally, the new electrochemical assay allowed to investigate the mechanisms of two different proteasome inhibitor drugs, bortezomib and oprozomib, underlying the applicability of the assay for understanding proteasome inhibitor action.

Preclinical comparison of proteasome and ubiquitin E1 enzyme inhibitors in cutaneous squamous cell carcinoma: the identification of mechanisms of differential sensitivity.

Proteasome inhibitors have distinct properties and the biochemical consequences of suppressing ubiquitin E1 enzymes and the proteasome differ. We compared the effects of the proteasome inhibitors bortezomib, ixazomib and carfilzomib and the ubiquitin E1 enzyme inhibitor MLN7243/TAK-243 on cell viability and cell death in normal keratinocytes and cutaneous squamous cell carcinoma (cSCC) cell lines. The effects of both a pulse of treatment and more extended incubation were investigated. This is relevant to directly-delivered therapy (topical treatment/intratumoral injection) where the time of exposure can be controlled and a short exposure may better reflect systemically-delivered inhibitor pharmacokinetics. These agents can selectively kill cSCC cells but there are variations in the pattern of cSCC cell line sensitivity/resistance. Variations in the responses to proteasome inhibitors are associated with differences in the specificity of the inhibitors for the three proteolytic activities of the proteasome. There is greater selectivity for killing cSCC cells compared to normal keratinocytes with a pulse of proteasome inhibitor treatment than with a more extended exposure. We provide evidence that c-MYC-dependent NOXA upregulation confers susceptibility to a short incubation with proteasome inhibitors by priming cSCC cells for rapid BAK-dependent death. We observed that bortezomib-resistant cSCC cells can be sensitive to MLN7243-induced death. Low expression of the ubiquitin E1 UBA1/UBE1 participates in conferring susceptibility to MLN7243 by increasing sensitivity to MLN7243-mediated attenuation of ubiquitination. This study supports further investigation of the potential of proteasome and ubiquitin E1 inhibition for cSCC therapy. Direct delivery of inhibitors could facilitate adequate exposure of skin cancers.

Anti-cachectic effect of Antrodia cinnamomea extract in lung tumor-bearing mice under chemotherapy.

Skeletal muscle atrophy, the most characteristic feature of cancer cachexia, often occurs in patients with cancer undergoing chemotherapy. Antrodia cinnamomea (AC) a widely used edible medical fungus, exhibits hepatoprotective, anti-inflammatory and anticancer activities. In this study, we investigated whether combined treatment with the ethonolic extract of AC ameliorates cachexia symptoms, especially muscle wasting, in lung tumor-bearing mice treated with chemotherapy. Our results revealed that gemcitabine and cisplatin-induced severe body weight loss and skeletal muscle atrophy in the mice with cancer were greatly attenuated after AC extract administration. The protection may be attributed to the inhibition of skeletal muscle proteolysis by suppressing myostatin and activin release, muscle wasting-related FoxO3/MuRF-1/MAFbx signaling, proteasomal enzyme activity, and pro-inflammatory cytokine production. A significant decrease in insulin-like growth factor 1 (IGF-1) expression and formation was observed in the atrophying muscle of the conventional chemotherapy treatment group (CGC), and this decrease was markedly reversed by AC treatment. Additionally, the anorexia, intestinal injury and dysfunction that occurred in the CGC group were mitigated by AC extract. Taken together, these results demonstrated that the AC extract has a protective effect against chemotherapy-induced muscle atrophy mainly by attenuating muscle proteolysis, pro-inflammatory cytokine production, and anorexia, and activating IGF-1-dependent protein synthesis.

Activation of the Nrf2 signaling pathway and neuroprotection of nigral dopaminergic neurons by a novel synthetic compound KMS99220

The transcription factor Nrf2 is known to induce gene expression of antioxidant enzymes and proteasome subunits. Because both oxidative stress and protein aggregation have damaging effects on neurons, activation of the Nrf2 signaling should be beneficial against neurodegeneration. In this study, we report a novel synthetic morpholine-containing chalcone KMS99220 that confers neuroprotection. It showed high binding affinity to the Nrf2 inhibitory protein Keap-1 and increased nuclear translocation of Nrf2 and gene expression of the antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and the catalytic and modifier subunits of glutamate-cysteine ligase in dopaminergic CATH.a cells. KMS99220 also increased expression of the proteasome subunits PSMB5, PSMB7, PSMB8 and PSMA1, and the respective chymotrypsin and trypsin-like proteasomal enzyme activities, and reduced α-synuclein aggregate in GFP-α-syn A53T-overexpressing cells. KMS99220 exhibited a favorable pharmacokinetic profile with excellent bioavailability and metabolic stability, did not interfere with activities of the cytochrome p450 isotypes, and showed no apparent in vivo toxicity when administered up to 2000 mg/kg. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, oral administration of KMS99220 prevented degeneration of the nigral dopaminergic neurons, induced the Nrf2 target genes, and effectively prevented the associated motor deficits. These results suggest KMS99220 as a potential candidate for therapy against Parkinson's disease.