20S proteasome

Epoxomicin exhibits strong cytotoxic effects on every tested cell type. Epoxomicin has been shown to inhibit the growth of B16-F10, HCT116, Moser, P388, and K562 cells, with IC50 values of 0.002 μg/mL, 0.005 μg/mL, 0.044 μg/mL, 0.002 μg/mL, and 0.037 μg/mL[1].
Epoxomicin possesses antiproliferative activity against EL4 lymphoma cells, with an IC50 of 4 nM[2].

Epoxomicin (0.063-1 mg/kg; intraperitoneal injection; once daily; for 9 days; male BDFX mice) treatment has a strong antitumor effect, with a minimum dosage of 0.13 mg/kg/day being effective[1].
Epoxomicin also potently blocks in vivo inflammation in the murine ear edema assay and effectively inhibits NF-κB activation in vitro[3].
Epoxomicin is given intraperitoneally over a two-week period to adult rats. The animals showed signs of progressive Parkinsonism, including bradykinesia, rigidity, tremor, and abnormal posture, after a latency of one to two weeks. Postmortem examinations reveal dopaminergic cell death with apoptosis and striatal dopamine depletion in the substantia nigra pars compacta[4].

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Epoxomicin Potently Reduces CS in Vivo. [1]
CS is an inflammatory response to certain classes of chemical compounds and haptens (30). Based on inhibition of NF-κB activation in cell culture, we hypothesized that epoxomicin would have antiinflammatory activities in vivo. To test this hypothesis, epoxomicin was evaluated in the picrylchloride mouse model of CS. Mice immunized with picrylchloride were challenged after 6 days by application of picrylchloride on their ears. Ear thickness measurements were made at 0 and 24 hr after picrylchloride ear challenge. As shown in Fig. 5, daily treatment with epoxomicin at a nontoxic dose of 0.58 mg/kg per day reduced the CS response by 44% relative to the control group of mice treated with vehicle alone. Because the hapten can elicit a nonspecific, irritation-related inflammatory response (31), we explored the effects of epoxomicin on skin irritation-mediated inflammation by using nonimmunized mice. In a second experiment, mice were pretreated with epoxomicin at a dose five times higher than that used previously to test the idea that a single injection of the drug could reduce inflammation in response to picrylchloride ear challenge. Epoxomicin administered at 2.9 mg/kg potently inhibited the irritant-associated inflammatory response by 95% when ear edema measurements were made 24 hr postchallenge (Fig. 5).

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Malaria continues to be a major global health problem, but only a limited arsenal of effective drugs is available. None of the antimalarial compounds commonly used clinically kill mature gametocytes, which is the form of the parasite that is responsible for malaria transmission. The parasite that causes the most virulent human malaria, Plasmodium falciparum, has a 48-h asexual cycle, while complete sexual differentiation takes 10 to 12 days. Once mature, stage V gametocytes circulate in the peripheral blood and can be transmitted for more than a week. Consequently, if chemotherapy does not eliminate gametocytes, an individual continues to be infectious for several weeks after the clearance of asexual parasites. The work reported here demonstrates that nanomolar concentrations of the proteasome inhibitor epoxomicin effectively kill all stages of intraerythrocytic parasites but do not affect the viability of human or mouse cell lines. Twenty-four hours after treatment with 100 nM epoxomicin, the total parasitemia decreased by 78%, asexual parasites decreased by 86%, and gametocytes decreased by 77%. Seventy-two hours after treatment, no viable parasites remained in the 100 or 10 nM treatment group. Epoxomicin also blocked oocyst production in the mosquito midgut. In contrast, the cysteine protease inhibitors epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester and N-acetyl-L-leucyl-L-leucyl-L-methioninal blocked hemoglobin digestion in early gametocytes but had no effect on later stages. Moreover, once the cysteine protease inhibitor was removed, sexual differentiation resumed. These findings provide strong support for the further development of proteasome inhibitors as antimalaria agents that are effective against asexual, sexual, and mosquito midgut stages of P. falciparum [5].

In assay solutions for proteasome inhibition assays, peptide-AMC substrates (5 μM Suc-LLVY-AMC, 5 μM Z-LLE-AMC, and 5 μM Boc-LRR-AMC) and epopoximin in DMSO are added at a final DMSO concentration of 1%. The assay buffer that is utilized is as follows: pH 8.0/0.5 mM EDTA with 20 mM Tris-HCl (plus 0.035% SDS for Z-LLE-AMC and Suc-LLVY-AMC assays). After adding the bovine red blood cell proteasome to the assay buffer containing substrates and doxomicin at a final volume of 100 μL at room temperature (23 °C), the fluorescence emission is immediately measured at 460 nm (λex, 360 nM) using a Cytofluor fluorescence plate reader for 50 minutes.

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Purification of epoxomicin-Binding Proteins.[1]
Ten liters of EL4 cells (106cells/ml) was harvested and resuspended in 50 ml of RPMI 1640 medium containing 10% FBS. Epoxomicin-biotin was added to a final concentration of 8 μM, and cells were incubated at 37°C for 4 hr. Cells were harvested and homogenized by using a Powergen homogenizer in lysis buffer (25 mM Hepes, pH 7.4/5 mM EGTA/50 mM NaF) plus protease inhibitors (10 μg/ml leupeptin, pepstatin, and soybean trypsin inhibitors and 1 mM PMSF). The high-speed (100,000 × g) supernatant was loaded onto a 1-ml streptavidin-agarose column to remove endogenous biotinylated proteins. The flow-through fraction then was incubated for 10 min with 50 ml of DE52 beads preequilibrated with lysis buffer, washed twice with 50 ml of lysis buffer containing 0.1 M NaCl, and eluted with 50 ml of lysis buffer containing 0.3 M NaCl. SDS was added to the eluant at a final concentration of 0.5%, boiled for 10 min, and diluted 2.5-fold by using lysis buffer. The diluted solution was loaded onto a 0.4-ml NeutrAvidin agarose column. The flow-through fraction was collected and reloaded onto the same column three times. After extensive washes, epoxomicin-biotin-binding proteins were eluted by boiling the NeutrAvidin agarose in 0.4 ml of 1× SDS sample buffer. The purified protein complexes were separated by SDS/PAGE, excised, and identified by the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University) by using LCQ (MS/MS) and automated Edman degradation of internal tryptic peptides.

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Enzyme Kinetic Assays.[1]
For proteasome inhibition assays, peptide-AMC substrates (5 μM Suc-LLVY-AMC, 5 μM Z-LLE-AMC, and 5 μM Boc-LRR-AMC) and inhibitors in DMSO were added to assay solutions at a final DMSO concentration of 1%. The following assay buffer was used: 20 mM Tris⋅HCl, pH 8.0/0.5 mM EDTA (plus 0.035% SDS for Suc-LLVY-AMC and Z-LLE-AMC assays). Bovine red blood cell proteasome was added to the assay buffer containing substrates and inhibitors at a final volume of 100 μl at room temperature (23°) in a Dynex Microfluor II 96-well plate and the fluorescence emission immediately was measured at 460 nm (λex, 360 nM) by using a Cytofluor fluorescence plate reader for 50 min. kobs/[I] values were obtained using kaleidagraph by nonlinear least-squares fit of the data to the following equation: fluorescence = vst + [(vo − vs)/kobs][1 − exp(−kobst)], where vo and vs are the initial and final velocities, respectively, and kobs is the reaction rate constant. Dilutions of bovine erythrocyte 20S proteasome (2.5 mg/ml) were as follows: 1:1,200 final dilution for Suc-LLVY-AMC activity, 1:3,000 for Z-LLE-AMC, and 1:800 for Boc-LRR-AMC. Inhibition reactions were performed as described previously. For calpain inhibition assays, the enzyme was used at 1 unit/ml, and Suc-LLVY-AMC was used at a final concentration of 10 μM in assay buffer containing 20 mM Tris, pH 8.0/1 mM CaCl2/2 mM DTT. Cathepsin B was used at a concentration of 0.005 unit/ml in 100 mM sodium acetate/5 mM EDTA, pH 5.5, and cathepsin substrate III was used as substrate at 40 μM. Kinetic assays were performed as described for the proteasome.

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EMSAs.[1]
EMSAs were performed as described. In brief, consensus DNA-binding oligonucleotide sequences for transcription factor NF-κB were labeled with [γ-32P]ATP and incubated with equal amounts of nuclear lysates. Protein–DNA complexes were separated on 4% polyacrylamide gels under nondenaturing and nonreducing conditions. The gels were dried and exposed to a PhosphorImaging screen for quantitation of radioactivity in retarded bands. Results are representative of experiments performed at least twice.

Cytotoxicity assays.[5]
Cells from a mouse fibroblast line, NIH-3T3, or a human alveolar basal epithelial cell line, A549 (ATCC) (1 × 104 to 5 × 104 cells per well in a 96-well plate), were incubated with serial dilutions of epoxomicin (32 to 2,000 nM) or carrier (DMSO) alone in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. The concentration of DMSO was kept below 1%, which did not affect cell viability. Forty-eight hours after the addition of drug, cell viability was assayed using the Cell Titer 96 aqueous nonradioactive cell proliferation assay according to the manufacturer's instructions (http://www.promega.com/enotes/applications/0004/ap0017.htm). The assay uses a soluble tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), that is reduced by viable cells to fromazan, which can be measured by absorbance at 492 nM.

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For IκB and ubiquitin Western blot analysis, 60% confluent HeLa monolayers were treated with 10 μM epoxomicin or Z-LLL-H for 2 hr. Tumor necrosis factor α (TNF-α) (10 ng/ml) was added to one set of the drug-treated plates and alone to a separate dish of cells. Cells were harvested after 15 min. p53 stabilization was analyzed by immunoprecipitating with mouse anti-p53 antisera from HUVECs treated with DMSO, 100 nM epoxomicin, or 5 μM Z-LLL-H for 6 hr followed by immunoblot analysis with rabbit anti-p53 antisera. For electrophoretic mobility-shift assays (EMSAs), epoxomicin was added to HeLa cells in duplicate and TNF-α (10 ng/ml) was added to one set of the drug-treated plates and also alone to a separate dish of cells. Cells were harvested after 1 hr, and nuclear lysates were prepared as described.

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After a pretreatment of vehicle, losartan (10μM), epoxomicin (10μM), or vehicle control, LECs were treated for a full day with either saline or Ang II (100μM).

BALB/c mice
2.9 mg/kg
intraperitoneal injection

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Assay for Contact Sensitivity (CS). [1]
CS and irritant-response assays to picrylchloride (a generous gift of P. Asekanse, Yale University) challenge were performed essentially as described, with slight modifications. In brief, for the CS assay, mice were injected i.p. daily for 6 days with vehicle or epoxomicin (0.58 mg/kg body weight) solubilized in 10% DMSO/PBS. Six days after abdomen immunization with picrylchloride, ear thickness measurements (0 hr) of both ears were made in triplicate with an engineer’s micrometer (Peacock dial thickness gauge; Ozaki Manufacturing, Tokyo). Mice subsequently were challenged on both ear lobes by application of 15 μl of a 0.8% solution of picrylchloride (solubilized in high-grade extra virgin olive oil). Ear swelling measurements again were made 24 hr post-ear challenge. In a second assay, elicitation of inflammatory response to the nonspecific vascular activation and permeability effects of picrylchloride (irritant response) were determined by using two groups of four nonimmunized mice. The 0-hr ear thickness measurements were made, a single high-dose injection of epoxomicin (2.9 mg/kg) was delivered i.p. to one group, and the control group was treated with vehicle. Ear thickness was measured 24 hr post-ear challenge.

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Plasmodium falciparum gametogenesis and mosquito feed assay. [1]
Cultures of P. falciparum parasites (strain 3D7) containing mature gametocytes were incubated for 1 h in the presence of ALLN, epoxomicin, or DMSO alone. To assay gametogenesis and exflagellation, an aliquot (0.5 ml) of the test cultures was pelleted and resuspended in emergence medium (10 μM xanthurenic acid, 1.67 mg ml−1 glucose, 8 mg ml−1 NaCl, 8 mM Tris-Cl [pH 8.2]). After a 10-min incubation at room temperature, parasite morphology and the number of exflagellating males was evaluated at a ×400 magnification. To evaluate oocyst production, an aliquot (1.2 ml) of the test cultures was pelleted onto 125-μl packed erythrocytes. The supernatant was replaced with 120 μl of normal human serum containing active complement, mixed, and introduced into a water-jacketed membrane feeder maintained at 37°C. Anopheles stephensi (SxK Nij.) mosquitoes were allowed to gorge for 10 min and then were grown for 7 more days at 25 ± 1°C and 80% ± 10% humidity. The midguts then were dissected and stained in 1.0% mercurochrome to visualize the P. falciparum oocysts.

[1]. Proc Natl Acad Sci U S A . 1999 Aug 31;96(18):10403-8.

[2]. Bioorg Med Chem Lett . 1999 Dec 6;9(23):3335-40.

[3]. J Immunol . 2000 Jun 15;164(12):6147-57.

[4]. Vet Parasitol . 2010 Jan 20;167(1):19-27.

[5]. Antimicrob Agents Chemother . 2009 Oct;53(10):4080-5.

Epoxomicin is a tripeptide consisting of an Ile-Ile-Thr-NH2 sequence N-substituted on the threonamide amidic nitrogen with a (2S)-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl group and with acetyl and methyl groups on the nitrogen of the isoleucine residue distal to the threonamide; a naturally occurring selective proteasome inhibitor with anti-inflammatory activity. It has a role as a proteasome inhibitor. It is a member of morpholines and a tripeptide.

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The proteasome regulates cellular processes as diverse as cell cycle progression and NF-kappaB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 microM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-kappaB activation in vitro and potently blocks in vivo inflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in various in vivo and in vitro systems. [1]

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While two structurally related epoxyketone-containing antitumor natural products, epoxomicin and eponemycin, share the proteasome as a common intracellular target, they differ in their antiproliferative activity, proteasome subunit binding specificity, and rates of proteasome inhibition. As a first step towards understanding such differences and developing novel proteasome subunit-specific inhibitors, we report here the synthesis and characterization of epoxomicin/dihydroeponemycin chimerae. [2]

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The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5-1 microM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89-168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5-1 microM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.[3]

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Epoxomicin potently and irreversibly inhibits the catalytic activity of proteasomal subunits. Treatment of proliferating cells with epoxomicin results in cell death through accumulation of ubiquinated proteins. Thus, epoxomicin has been proposed as a potential anti-cancer drug. In the present study, the inhibitory effects of epoxomicin on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of epoxomicin on the in vivo growth of Babesia microti was also assessed. The in vitro growth of five Babesia species that were tested was significantly inhibited (P<0.05) by nanomolar concentrations of epoxomicin (IC(50) values=21.4+/-0.2, 4+/-0.1, 39.5+/-0.1, 9.7+/-0.3, and 21.1+/-0.1nM for Babesia bovis, Babesia bigemina, Babesia ovata, Babesia caballi, and Babesia equi, respectively). Epoxomicin IC(50) values for Babesia parasites were low when compared with diminazene aceturate and tetracycline hydrochloride. Combinations of epoxomicin with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis, B. bigemina, and B. caballi. In B. microti-infected mice, epoxomicin caused significant (P<0.05) inhibition of the growth of B. microti at the non-toxic doses of 0.05 and 0.5mg/kg BW relative to control groups. Therefore, epoxomicin might be used for treatment of babesiosis.[4]

C28H50N4O7

554.72

554.367

C, 60.63; H, 9.09; N, 10.10; O, 20.19

134381-21-8

11226684

White to off-white solid powder

1.1±0.1 g/cm3

795.7±60.0 °C at 760 mmHg

107-109ºC

435.0±32.9 °C

0.0±6.3 mmHg at 25°C

1.504

3.32

4

7

16

39

895

8

O=C([C@@H](NC([C@H]([C@@H](C)O)NC([C@@H](NC([C@H]([C@@H](C)CC)N(C)C(C)=O)=O)[C@@H](C)CC)=O)=O)CC(C)C)[C@@]1(C)CO1

DOGIDQKFVLKMLQ-JTHVHQAWSA-N

InChI=1S/C28H50N4O7/c1-11-16(5)21(30-27(38)23(17(6)12-2)32(10)19(8)34)25(36)31-22(18(7)33)26(37)29-20(13-15(3)4)24(35)28(9)14-39-28/h15-18,20-23,33H,11-14H2,1-10H3,(H,29,37)(H,30,38)(H,31,36)/t16-,17-,18+,20-,21-,22-,23-,28+/m0/s1

(2S,3S)-2-[[(2S,3S)-2-[acetyl(methyl)amino]-3-methylpentanoyl]amino]-N-[(2S,3R)-3-hydroxy-1-[[(2S)-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl]amino]-1-oxobutan-2-yl]-3-methylpentanamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.51 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.51 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.51 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)