Anthracyclines: biosynthesis, engineering and clinical applications.

Covering: January 1995 to June 2021Anthracyclines are glycosylated microbial natural products that harbour potent antiproliferative activities. Doxorubicin has been widely used as an anticancer agent in the clinic for several decades, but its use is restricted due to severe side-effects such as cardiotoxicity. Recent studies into the mode-of-action of anthracyclines have revealed that effective cardiotoxicity-free anthracyclines can be generated by focusing on histone eviction activity, instead of canonical topoisomerase II poisoning leading to double strand breaks in DNA. These developments have coincided with an increased understanding of the biosynthesis of anthracyclines, which has allowed generation of novel compound libraries by metabolic engineering and combinatorial biosynthesis. Coupled to the continued discovery of new congeners from rare Actinobacteria, a better understanding of the biology of Streptomyces and improved production methodologies, the stage is set for the development of novel anthracyclines that can finally surpass doxorubicin at the forefront of cancer chemotherapy.

The identification of the anthracycline aclarubicin as an effective cytotoxic agent for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find novel treatment options for PDAC. Here, we tested a series of conventional chemotherapeutics together with anthracycline compounds as single agents or in combination, determining their effectivity against established commercial and patient-derived, low-passage PDAC cell lines. Proliferation and colony formation assays were performed to determine the anticancer activity of anthracyclines; aclarubicin and doxorubicin, on commercial and patient-derived, low-passage PDAC cell lines. In addition, the effect of standard-of-care drugs gemcitabine and individual components of FOLFIRINOX were also investigated. To evaluate which mechanisms of cell death were involved in drug response, cleavage of poly(ADP-ribose)polymerase was evaluated by western blot. Aclarubicin showed superior antitumor activity compared to other anthracyclines and standard of care drugs (gemcitabine and individual components of FOLFIRINOX) in a patient-derived, low-passage PDAC cell line and in commercial cell lines. Importantly, the combination of gemcitabine and aclarubicin showed a synergistic effect at a dose range where the single agents by themselves were ineffective. In parallel, evaluation of the antitumor activity of aclarubicin demonstrated an apoptotic effect in all PDAC cell lines. Aclarubicin is cytotoxic for commercial and patient-derived low-passage PDAC cell lines, at doses lower than peak serum concentrations for patient treatment. Our findings support a (re)consideration of aclarubicin as a backbone of new combination regimens for pancreatic cancer patients.

Synthetic (N,N-Dimethyl)doxorubicin Glycosyl Diastereomers to Dissect Modes of Action of Anthracycline Anticancer Drugs.

Anthracyclines are effective drugs in the treatment of various cancers, but their use comes with severe side effects. The archetypal anthracycline drug, doxorubicin, displays two molecular modes of action: DNA double-strand break formation (through topoisomerase IIα poisoning) and chromatin damage (via eviction of histones). These biological activities can be modulated and toxic side effects can be reduced by separating these two modes of action through alteration of the aminoglycoside moiety of doxorubicin. We herein report on the design, synthesis, and evaluation of a coherent set of configurational doxorubicin analogues featuring all possible stereoisomers of the 1,2-amino-alcohol characteristic for the doxorubicin 3-amino-2,3-dideoxyfucoside, each in nonsubstituted and N,N-dimethylated forms. The set of doxorubicin analogues was synthesized using appropriately protected 2,3,6-dideoxy-3-amino glycosyl donors, equipped with an alkynylbenzoate anomeric leaving group, and the doxorubicin aglycon acceptor. The majority of these glycosylations proceeded in a highly stereoselective manner to provide the desired axial α-linkage. We show that both stereochemistry of the 3-amine carbon and N-substitution state are critical for anthracycline cytotoxicity and generally improve cellular uptake. N,N-Dimethylepirubicin is identified as the most potent anthracycline that does not induce DNA damage while remaining cytotoxic.

Association between Salmonella infection and colon cancer: a nationwide registry-based cohort study.

Laboratory data increasingly suggest that Salmonella infection may contribute to colon cancer (CC) development. Here, we examined epidemiologically the potential risk of CC associated with salmonellosis in the human population. We conducted a population-based cohort study using four health registries in Denmark. Person-level demographic data of all residents were linked to laboratory-confirmed non-typhoidal salmonellosis and to CC diagnoses in 1994-2016. Hazard ratios (HRs) for CC (overall/proximal/distal) associated with reported salmonellosis were estimated using Cox proportional hazard models. Potential effects of serovar, age, sex, inflammatory bowel disease and follow-up time post-infection were also assessed. We found no increased risk of CC ≥1 year post-infection (HR 0.99; 95% confidence interval (CI) 0.88-1.13). When stratifying by serovar, there was a significantly increased risk of proximal CC ≥1 year post-infection with serovars other than Enteritidis and Typhimurium (HR 1.40; 95% CI 1.03-1.90). CC risk was significantly increased in the first year post-infection (HR 2.08; 95% CI 1.48-2.93). The association between salmonellosis and CC in the first year post-infection can be explained by increased stool testing around the time of CC diagnosis. The association between proximal CC and non-Enteritidis/non-Typhimurium serovars is unclear and warrants further investigation. Overall, this study provides epidemiological evidence that notified Salmonella infections do not contribute significantly to CC risk in the studied population.

Immunoproteasome Inhibitor-Doxorubicin Conjugates Target Multiple Myeloma Cells and Release Doxorubicin upon Low-Dose Photon Irradiation.

Proteasome inhibitors are established therapeutic agents for the treatment of hematological cancers, as are anthracyclines such as doxorubicin. We here present a new drug targeting approach that combines both drug classes into a single molecule. Doxorubicin was conjugated to an immunoproteasome-selective inhibitor via light-cleavable linkers, yielding peptide epoxyketone-doxorubicin prodrugs that remained selective and active toward immunoproteasomes. Upon cellular uptake and immunoproteasome inhibition, doxorubicin is released from the immunoproteasome inhibitor through photoirradiation. Multiple myeloma cells in this way take a double hit: immunoproteasome inhibition and doxorubicin-induced toxicity. Our strategy, which entails targeting of a cytotoxic agent, through a covalent enzyme inhibitor that is detrimental to tumor tissue in its own right, may find use in the search for improved anticancer drugs.

Occupational risk of salmonellosis and campylobacteriosis: a nationwide population-based registry study.

OBJECTIVES: Occupational exposure to animals and foods thereof is a poorly characterised risk factor for salmonellosis and campylobacteriosis, the main causes of bacterial gastroenteritis in the Western world. We performed a population-based registry study in the Netherlands to assess whether differences exist in the incidence of reported salmonellosis and campylobacteriosis cases among occupational groups, and whether they can be explained by differences in the magnitude of exposure to these pathogens, as defined by serology. METHODS: Person-level occupational data for all Dutch residents were linked to lab-confirmed salmonellosis and campylobacteriosis data, and to serological data from a previous national serosurvey. SIRs for salmonellosis and campylobacteriosis among occupational sectors and specific high-risk occupations were calculated based on the total employed population. Moreover, Salmonella and Campylobacter seroincidence rates were compared among sectors and high-risk occupations. RESULTS: Occupational exposure to live animals or manure and working in the sale of animal-derived food products were associated with significantly increased risks of salmonellosis (SIR 1.55-1.82) and campylobacteriosis (SIR 1.36-1.65). Moreover, incidences were significantly higher in specific industrial sectors, as well as healthcare and social work sectors. Mean seroincidence rates ranged from 1.28 to 2.30 infections/person-year for Campylobacter, and from 0.36 to 0.99 for Salmonella, with only slightly higher rates for people in high-risk occupations. CONCLUSIONS: Significant differences in reported salmonellosis and campylobacteriosis incidence exist among occupational sectors, with the highest incidence in those persons occupationally exposed to live animals. These differences are only partially reflected in the serology.

Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

Cowpox virus protein CPXV012 eludes CTLs by blocking ATP binding to TAP.

CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs.

Metabolic engineering of Streptomyces peucetius for biosynthesis of N,N-dimethylated anthracyclines.

Introduction: Daunorubicin and doxorubicin, two anthracycline polyketides produced by S. peucetius, are potent anticancer agents that are widely used in chemotherapy, despite severe side effects. Recent advances have highlighted the potential of producing improved derivatives with reduced side effects by incorporating l-rhodosamine, the N,N-dimethyl analogue of the native amino sugar moiety. Method: In this study, we aimed to produce N,N-dimethylated anthracyclines by engineering the doxorubicin biosynthetic pathway in the industrial Streptomyces peucetius strain G001. To achieve this, we introduced genes from the aclarubicin biosynthetic pathway encoding the sugar N-methyltransferases AclP and AknX2. Furthermore, the native gene for glycosyltransferase DnrS was replaced with genes encoding the aclarubicin glycosyltransferases AknS and AknT. Additionally, the gene for methylesterase RdmC from the rhodomycin biosynthetic pathway was introduced. Results: A new host was engineered successfully, whereby genes from the aclarubicin pathway were introduced and expressed. LC-MS/MS analysis of the engineered strains showed that dimethylated sugars were efficiently produced, and that these were incorporated ino the anthracycline biosynthetic pathway to produce the novel dimethylated anthracycline N,N-dimethyldaunorubicin. Further downstream tailoring steps catalysed by the cytochrome P450 monooxygenase DoxA exhibited limited efficacy with N,N-dimethylated substrates. This resulted in only low production levels of N,N-dimethyldaunorubicin and no N,N-dimethyldoxorubicin, most likely due to the low affinity of DoxA for dimethylated substrates. Discussion: S. peucetius G001 was engineered such as to produce N,N-dimethylated sugars, which were incorporated into the biosynthetic pathway. This allowed the successful production of N,N-dimethyldaunorubicin, an anticancer drug with reduced cytotoxicity. DoxA is the key enzyme that determines the efficiency of the biosynthesis of N,N-dimethylated anthracyclines, and engineering of this enzyme will be a major step forwards towards the efficient production of more N,N-dimethylated anthracyclines, including N,N-dimethyldoxorubicin. This study provides valuable insights into the biosynthesis of clinically relevant daunorubicin derivatives, highlighting the importance of combinatorial biosynthesis.

A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates.

gamma 1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like alpha- and beta-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349-360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829-6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8(+) T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related gamma 1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8(+) T cell recognition through HLA-A-, HLA-B-, and HLA-C-restricting alleles when expressed in target cells in vitro. The small (60-amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate gamma 1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8(+) T cell control over virus replicative foci.

Re-Exploring the Anthracycline Chemical Space for Better Anti-Cancer Compounds.

The anthracycline anti-cancer drugs are intensely used in the clinic to treat a wide variety of cancers. They generate DNA double strand breaks, but recently the induction of chromatin damage was introduced as another major determinant of anti-cancer activity. The combination of these two events results in their reported side effects. While our knowledge on the structure-activity relationship of anthracyclines has improved, many structural variations remain poorly explored. Therefore, we here report on the preparation of a diverse set of anthracyclines with variations within the sugar moiety, amine alkylation pattern, saccharide chain and aglycone. We assessed the cytotoxicity in vitro in relevant human cancer cell lines, and the capacity to induce DNA- and chromatin damage. This coherent set of data allowed us to deduce a few guidelines on anthracycline design, as well as discover novel, highly potent anthracyclines that may be better tolerated by patients.

Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy.

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to gamma-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes.

Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.

Spatially resolved sampling for untargeted metabolomics: A new tool for salivomics.

Saliva is a complex bodily fluid composed of metabolites secreted by major and minor glands, as well as by-products of host oral cells, oral bacteria, gingival crevicular fluid, and exogenous compounds. Major salivary glands include the paired parotid, submandibular, and sublingual glands. The secreted fluids of the salivary glands vary in composition, flow rate, site of release, and clearance suggesting that different types of saliva fulfill different functions and therefore can provide unique biological information. Consequently, for the comprehension of the functionality of the salivary components, spatially resolved investigations are warranted. To understand and comprehensively map the highly heterogeneous environment of the oral cavity, advanced spatial sampling techniques for metabolomics analysis are needed. Here, we present a systematic evaluation of collection devices for spatially resolved sampling aimed at untargeted metabolomics and propose a comprehensive and reproducible collection and analysis protocol for the spatially resolved analysis of the human oral metabolome.

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP.

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

A brief report on combination chemotherapy and anti-programmed death (ligand) 1 treatment in small-cell lung cancer: Did we choose the optimal chemotherapy backbone?

Extensive-stage small-cell lung cancer (ES-SCLC) is an aggressive cancer that remains very hard to treat. The life expectancy of a patient diagnosed with this disease has not changed over the past three decades. Recently, three large clinical studies showed a survival benefit by adding an anti-programmed death (ligand) 1 (PD-(L)1 antibody to the current chemotherapy regimen. Although significant and important, the benefit seems less than what has been achieved in patients with non-small-cell lung cancer treated with chemoimmunotherapy. A number of hypotheses have been explored to explain this discrepancy. Here, we hypothesise that the current chemotherapy backbone in ES-SCLC does not contain the optimal drugs to trigger immunogenic cell death and therefore does not induce a synergy between chemotherapy and immune checkpoint inhibitor therapy. Thereby, we advocate that doxorubicin treatment instead of etoposide should be reconsidered as standard-of-care (SoC) first-line treatment of SCLC.

A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export.

Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.

Detection of aberrant transcription of major histocompatibility complex class II antigen presentation genes in chronic lymphocytic leukaemia identifies HLA-DOA mRNA as a prognostic factor for survival.

In human B cells, effective major histocompatibility complex (MHC) class II-antigen presentation depends not only on MHC class II, but also on the invariant chain (CD74 or Ii), HLA-DM (DM) and HLA-DO (DO), the chaperones regulating the antigen loading process of MHC class II molecules. We analysed immediate ex vivo expression of HLA-DR (DR), CD74, DM and DO in B cell chronic lymphocytic leukaemia (B-CLL). Real-time reverse transcription polymerase chain reaction demonstrated a highly significant upregulation of DRA, CD74, DMB, DOA and DOB mRNA in purified malignant cells compared to B cells from healthy donors. The increased mRNA levels were not translated into enhanced protein levels but could reflect aberrant transcriptional regulation. Indeed, upregulation of DRA, DMB, DOA and DOB mRNA correlated with enhanced expression of class II transactivator (CIITA). In-depth analysis of the various CIITA transcripts demonstrated a significant increased activity of the interferon-gamma-inducible promoter CIITA-PIV in B-CLL. Comparison of the aberrant mRNA levels with clinical outcome identified DOA mRNA as a prognostic indicator for survival. Multivariate analysis revealed that the prognostic value of DOA mRNA was independent of the mutational status of the IGHV genes. Thus, aberrant transcription of DOA forms a novel and additional prognostic indicator for survival in B-CLL.

Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm.

The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.

A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo.

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx(3)L(3)VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.