Epoxomicin

Epoxomicin was originally isolated from the culture medium of an Actinomycetes strain based on its in vivo antitumor activity against murine B16 melanoma. Epoxomicin is a naturally occurring selective proteasome inhibitor with anti-inflammatory activity. ^[1] Epoxomicin primarily inhibits the activity of CTRL (chymotrypsin-like proteasome).

The novel α-epoxy ketone moiety of Epoxomicin forms covalent bonds with residues in particular catalytic subunits of the enzyme, disabling activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing behaviors of the proteasome were both inhibited by Epoxomicin as well (at 100 and 1,000-fold slower rates, respectively). The ubiquitin-proteasome pathway heavily regulates bone formation, and Epoxomicin was shown to increase both bone volume and bone formation rates in rodents.

Another study demonstrates that exposure to Epoxomicin and other proteasome inhibitors leads to dopaminergic cell death, producing a model of Parkinson's disease in vivo. Epoxomicin is an inhibitor of 20S Proteasome. ^[2]

References:
1. Meng, L; Mohan, R; Kwok, BH; Elofsson, M; Sin, N; Crews, CM (1999). "Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity". PNAS 96 (18): 10403–10408.
2. Epoxomicin, Santa Cruz Biotechnology.

Regulation of ubiquitin-proteasome and autophagy pathways after acute LPS and epoxomicin administration in mice.[Pubmed:24885455]

BMC Musculoskelet Disord. 2014 May 22;15:166.

BACKGROUND: The ubiquitin-proteasome pathway (UPP) is a major protein degradation pathway that is activated during sepsis and has been proposed as a therapeutic target for preventing skeletal muscle loss due to cachexia. Although several studies have investigated the modulation of proteasome activity in response to LPS administration, none have characterized the overall UPP response to LPS administration in the fate of proteasome inhibition. METHODS: Here, we determined the modulation pattern of the main key components of the UPP in the gastrocnemius (GAS) of mice during the acute phase of lipopolysaccharide (LPS)-mediated endotoxemia (7.5 mg/kg - 8 h) by measuring all three beta1, beta2 and beta5 activites of the 20S and 26S proteasomes, the levels of steady state polyubiquitinated proteins, mRNA levels of muscle ligases, as well as signaling pathways regulating the UPP. Another goal was to assess the effects of administration of a specific proteasome inhibitor (Epoxomicin, 0.5 mg/kg) on UPP response to sepsis. RESULTS: The acute phase of LPS-induced endotoxemia lowered GAS/body weight ratio and increased MuRF1 and MAFbx mRNA concomitantly to an activation of the pathways known to regulate their expression. Unexpectedly, we observed a decrease in all 20S and 26S proteasome activities measured in GAS, which might be related to oxidative stress, as oxidized proteins (carbonyl levels) increase with LPS. While significantly inhibiting 20S and 26S proteasome beta5 activities in heart and liver, Epoxomicin did not lower proteasome activity in GAS. However, the increase in mRNA expression of the muscle ligases MuRF1 and MAFbx were partially rescued without affecting the other investigated signaling pathways. LPS also strongly activated autophagy, which could explain the observed GAS atrophy with LPS-induced reduction of proteasome activity. CONCLUSIONS: Our results highlight an opposite regulation of UPP in the early hours of LPS-induced muscle atrophy by showing reduced proteasome activities and increased mRNA expression of muscle specific ligases. Furthermore, our data do not support any preventive effect of Epoxomicin in muscle atrophy due to acute cachexia since proteasome activities are not further repressed.

Epoxomicin Sensitizes Resistant Osteosarcoma Cells to TRAIL Induced Apoptosis.[Pubmed:25666501]

Anticancer Agents Med Chem. 2015;15(4):527-33.

Osteosarcoma (OS) is the second most common primary malign bone neoplasm after multiple myeloma. Despite systemic chemotherapy, OS may give rise to local recurrences and metastases. Resistance to chemotherapy is not rare and is likely to occur in a high number of patients. Novel therapeutic approaches are required in order to efficiently treat osteosarcoma. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and proteasome inhibitors (Epoxomicin, MG132, bortezomib) represent new promising approaches in cancer treatment. The aim of our study is to elucidate the effects of Epoxomicin alone or in combination with TRAIL in two TRAIL-resistant OS cell lines, Saos-2 and MG-63 namely. We determined the cytotoxic effects of Epoxomicin and/or TRAIL on these two types of OS cells using dimethylthiazolyl 2,5 diphenyltetrazolium bromide (MTT) test and measured apoptosis markers such as pro-apoptotic Bax levels and caspase-3, -8, -9 activities. We used TUNEL assay to demonstrate apoptosis. We investigated dose and time dependent survival rates of OS cells and determined LD50 doses of Epoxomicin and TRAIL on OS cell viability after 24, 48, and 72 hour incubations. Concurrent incubation with TRAIL and Epoxomicin for 24 hour significantly increased caspase-3, caspase-8, caspase-9 activities and Bax protein levels. Our study demonstrated that the combination of TRAIL with Epoxomicin enhances apoptosis, and overcomes TRAIL resistance, denoting promising results for OS therapy in the future.

Epoxomicin and Eponemycin Biosynthesis Involves gem-Dimethylation and an Acyl-CoA Dehydrogenase-Like Enzyme.[Pubmed:26789439]

Chembiochem. 2016 May 3;17(9):792-8.

The alpha',beta'-epoxyketone moiety of proteasome inhibitors confers high binding specificity to the N-terminal threonine in catalytic proteasome beta-subunits. We recently identified the Epoxomicin and eponemycin biosynthetic gene clusters and have now conducted isotope-enriched precursor feeding studies and comprehensive gene deletion experiments to shed further light on their biosynthetic pathways. Leucine and two methyl groups from S-adenosylmethionine were readily incorporated into the epoxyketone warhead, suggesting decarboxylation of the thioester intermediate. Formation of the alpha',beta'-epoxyketone is likely mediated by conserved acyl-CoA dehydrogenase-like enzymes, as indicated by complete loss of Epoxomicin and eponemycin production in the respective knockout mutants. Our results clarify crucial questions in the formation of epoxyketone compounds and lay the foundation for in vitro biochemical studies on the biosynthesis of this pharmaceutically important class of proteasome inhibitors.

From epoxomicin to carfilzomib: chemistry, biology, and medical outcomes.[Pubmed:23575525]

Nat Prod Rep. 2013 May;30(5):600-4.

The initial enthusiasm following the discovery of a pharmacologically active natural product is often fleeting due to the poor prospects for its ultimate clinical application. Despite this, the ever-changing landscape of modern biology has a constant need for molecular probes that can aid in our understanding of biological processes. After its initial discovery by Bristol-Myers Squibb as a microbial anti-tumor natural product, Epoxomicin was deemed unfit for development due to its peptide structure and potentially labile epoxyketone pharmacophore. Despite its drawbacks, Epoxomicin's pharmacophore was found to provide unprecedented selectivity for the proteasome. Epoxomicin also served as a scaffold for the generation of a synthetic tetrapeptide epoxyketone with improved activity, YU-101, which became the parent lead compound of carfilzomib (Kyprolis), the recently approved therapeutic agent for multiple myeloma. In this era of rational drug design and high-throughput screening, the prospects for turning an active natural product into an approved therapy are often slim. However, by understanding the journey that began with the discovery of Epoxomicin and ended with the successful use of carfilzomib in the clinic, we may find new insights into the keys for success in natural product-based drug discovery.

Epoxomicin (BU-4061T) is an epoxyketone-containing natural product and a potent, selective and irreversible proteasome inhibitor. Epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome and potently inhibits primarily the chymotrypsin-like activity. Epoxomicin can cross the blood-brain barrier. Epoxomicin has strongly antitumor and anti-inflammatory activity.

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