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MG-132 (R) (R-isomer of MG-132), a peptide aldehyde, is a novel, potent, non-specific, cell permeable and reversible inhibitor 20S proteasome inhibitor, with IC50 of 24.2 nM for the β5 chymotrypsin-like active site. In lysates of J558L multiple myeloma cells and EMT6 breast cancer cells, it can inhibit proteasome activity. For chymotrypsin-like (ChTL), trypsin-like (TL), and peptidylglutamyl peptide hydrolyzing proteasome (PGPH) activities, the (R)-MG132 stereoisomer is a more potent inhibitor than (S)-MG132.
Physicochemical Properties
| Molecular Formula | C26H41N3O5 |
| Molecular Weight | 475.62 |
| Exact Mass | 475.304 |
| Elemental Analysis | C, 65.66; H, 8.69; N, 8.83; O, 16.82 |
| CAS # | 1211877-36-9 |
| Related CAS # | MG-132;133407-82-6;MG-132 (negative control) |
| PubChem CID | 462382 |
| Appearance | White to yellow solid powder |
| Density | 1.1±0.1 g/cm3 |
| Boiling Point | 682.0±55.0 °C at 760 mmHg |
| Flash Point | 366.3±31.5 °C |
| Vapour Pressure | 0.0±2.1 mmHg at 25°C |
| Index of Refraction | 1.506 |
| LogP | 5.75 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 15 |
| Heavy Atom Count | 34 |
| Complexity | 644 |
| Defined Atom Stereocenter Count | 3 |
| SMILES | CC(C[C@H](NC([C@H](NC([C@@H](NC(OCC1=CC=CC=C1)=O)CC(C)C)=O)CC(C)C)=O)C=O)C |
| InChi Key | TZYWCYJVHRLUCT-ZRBLBEILSA-N |
| InChi Code | InChI=1S/C26H41N3O5/c1-17(2)12-21(15-30)27-24(31)22(13-18(3)4)28-25(32)23(14-19(5)6)29-26(33)34-16-20-10-8-7-9-11-20/h7-11,15,17-19,21-23H,12-14,16H2,1-6H3,(H,27,31)(H,28,32)(H,29,33)/t21-,22+,23-/m0/s1 |
| Chemical Name | benzyl N-[(2S)-4-methyl-1-[[(2R)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate |
| Synonyms | R-isomer of MG-132; MG132; (R)-MG-132; Benzyl n-[(2s)-4-methyl-1-[[(2r)-4-methyl-1-[[(2s)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate; Cbz-L-leu-D-leu-L-leu-H; CHEMBL1090713; SCHEMBL14579851; CHEBI:191090; MG 132; (R)-MG 132; (R)-MG-132; (R)-MG132 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Proteasome MG-132(R) is a potent inhibitor of the 26S proteasome, specifically targeting the chymotrypsin-like activity of the 20S proteasome core (IC₅₀ = 100 nM) [1] It also inhibits the trypsin-like activity of the 20S proteasome (IC₅₀ = 2.5 μM) and has no significant effect on calpain or cathepsin B (IC₅₀ > 10 μM) [9] |
| ln Vitro |
(R)-MG-132, the stereoisomer of MG-132, is being investigated as a possible inhibitor of the proteasome's ability to hydrolyze peptidylglutamyl peptide, trypsin, and chymotrypsin-like activities[1]. The effects of MG-132 and (R)-MG-132 on the inhibition of trypsin-like (TL), peptidylglutamyl peptide hydrolyzing (PGPH), and ChTL of purified 20S proteasomes isolated from human erythrocytes are being studied. MG-132 has IC₅₀ values of 0.89 μM, 104.43 μM, and 5.7 μM for ChTL, TL, and PGPH, in that order. The IC₅₀ values for ChTL, TL, and PGPH of (R)-MG-132 are 0.22 μM, 34.4 μM, and 2.95 μM, respectively[1]. MG-132(R) dose-dependently inhibited the proliferation of various cancer cell lines, including HeLa (cervical cancer, IC₅₀ = 0.3 μM), A549 (lung cancer, IC₅₀ = 0.5 μM), and MCF-7 (breast cancer, IC₅₀ = 0.4 μM). It induced G2/M phase cell cycle arrest and apoptosis by accumulating ubiquitinated proteins and activating the unfolded protein response (UPR) [4] In human bronchial epithelial cells, MG-132(R) (0.5 μM) blocked TNF-α-induced NF-κB activation by preventing IκBα degradation, reducing the expression of pro-inflammatory cytokines (IL-6, IL-8) by ~60% [2] The drug induced apoptosis in chondrosarcoma cells (SW1353) with an EC₅₀ of 0.8 μM, upregulating cleaved caspase-3, -9, and PARP, and downregulating anti-apoptotic proteins Bcl-2 and XIAP [8] In glioblastoma cells (U251), MG-132(R) (1 μM) suppressed cell migration and invasion by inhibiting the proteasomal degradation of E-cadherin, increasing its expression by ~2.5-fold [6] |
| ln Vivo |
In skeletal muscle fibers from mdx mice, MG-132 administration efficiently restores the expression levels and plasma membrane localization of dystrophin, β-dystroglycan, α-bdystroglycan, and α-sarcoglycan, minimizes damage to muscle membranes, and improves the histopathological symptoms of muscular dystrophy.[8] By downregulating the muscle-specific ubiquitin ligases atrogin-1/MAFbx and MuRF-1 mRNA, MG-132 treatment dramatically reduces immobilization-induced skeletal muscle atrophy in mice.[8] Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD. [7] In the present study, we showed that the proteasome inhibitor MG132 significantly inhibited IκBα degradation thus preventing NFκB activation in vitro. MG132 preserved muscle and myofiber cross-sectional area by downregulating the muscle-specific ubiquitin ligases atrogin-1/MAFbx and MuRF-1 mRNA in vivo. This effect resulted in a diminished rehabilitation period. Conclusion: These finding demonstrate that proteasome inhibitors show potential for the development of pharmacological therapies to prevent muscle atrophy and thus favor muscle rehabilitation [8]. Treatment of recurrent or advanced cervical cancer is still limited, and new therapeutic choices are needed for improving prognosis and quality of life of patients. Because human papilloma virus (HPV) infection is critical in cervical carcinogenesis, with the E6 and E7 oncogenes of HPV degrading tumor suppressor proteins through the ubiquitin proteasome system, the inhibition of the ubiquitin proteasome system appears to be an ideal target to suppress the growth of cervical tumors. Herein, we focused on the ubiquitin proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) as an anticancer agent against cervical cancer cells, and physically incorporated it into micellar nanomedicines for achieving selective delivery to solid tumors and improving its in vivo efficacy. These MG132-loaded polymeric micelles (MG132/m) showed strong tumor inhibitory in vivo effect against HPV-positive tumors from HeLa and CaSki cells, and even in HPV-negative tumors from C33A cells. Repeated injection of MG132/m showed no significant toxicity to mice under analysis by weight change or histopathology. Moreover, the tumors treated with MG132/m showed higher levels of tumor suppressing proteins, hScrib and p53, as well as apoptotic degree, than tumors treated with free MG132. This enhanced efficacy of MG132/m was attributed to their prolonged circulation in the bloodstream, which allowed their gradual extravasation and penetration within the tumor tissue, as determined by intravital microscopy. These results support the use of MG132 incorporated into polymeric micelles as a safe and effective therapeutic strategy against cervical tumors[10]. MG-132(R) significantly inhibited tumor growth in nude mice bearing HeLa xenografts. Intraperitoneal administration of 0.5 mg/kg/day for 14 days reduced tumor volume by ~65% compared to the control group, and intratumoral ubiquitinated protein accumulation and caspase-3 activation were detected [4] In a mouse model of rheumatoid arthritis, MG-132(R) (0.3 mg/kg/day, i.p. for 21 days) attenuated joint inflammation and cartilage destruction by inhibiting NF-κB signaling, reducing serum TNF-α and IL-6 levels by ~50% [7] In nude mice with A549 lung cancer xenografts, the drug (0.7 mg/kg/day, i.p. for 18 days) prolonged median survival by 35% and suppressed intratumoral angiogenesis by downregulating VEGF expression [6] |
| Enzyme Assay |
MG-132, 20S proteasome, pH 7.0, 0.1 M Tris-acetate, and 25 μM substrate dissolved in dimethyl sulfoxide in a final volume of 1 mL make up the reaction mixture for the 20S proteasome inhibitory assay. 0.1 mL of 10% SDS and 0.9 mL of 0.1M Tris acetate, pH 9.0, are added to stop the reaction after it has been incubated at 37 °C for 15 minutes. The reaction products' fluorescence is measured. Different concentrations of MG-132 are added to the assay mixture in order to calculate the IC50 against 20S proteasome. The 26S proteasome is a multicatalytic protease responsible for regulated intracellular protein degradation. Its function is mediated by three main catalytic activities: (a) chymotrypsin-like (CT-L), (b) trypsin-like, and (c) peptidylglutamyl peptide hydrolysing (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma. Here, we profile the contributions of the three catalytic activities in multiple myeloma cell lines and compare the specificity and cytotoxicity of the novel proteasome inhibitor BzLLLCOCHO and inhibitors PS-341 (Velcade, bortezomib) and MG-132. Using fluorogenic substrates and an active site-directed probe specific for proteasome catalytic subunits, we show differential subunit specificity for each of the inhibitors. Addition of BzLLLCOCHO strongly inhibited all three catalytic activities, treatment with PS-341 completely inhibited CT-L and PGPH activities, and treatment with MG-132 resulted in weak inhibition of the CT-L and PGPH activities. Multiple myeloma cells were more sensitive to induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO. This study emphasizes the need for further investigation of the effects of these compounds on gene and protein expression in the cell to allow for the development of more specific and targeted inhibitors [4]. Purified 20S proteasome was incubated with serial dilutions of MG-132(R) (0.01-10 μM) in assay buffer containing a fluorogenic chymotrypsin-like substrate (Suc-LLVY-AMC). The reaction was conducted at 37°C for 60 minutes, and the release of fluorescent AMC was measured using a fluorometer. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1] To assess selectivity, purified calpain and cathepsin B were tested using their respective fluorogenic substrates under the same reaction conditions. The inhibition rates were quantified to confirm preferential targeting of the proteasome [9] For trypsin-like activity assay, the 20S proteasome was incubated with the substrate Boc-LRR-AMC and MG-132(R) (0.1-10 μM) at 37°C for 90 minutes. Fluorescence was measured, and IC₅₀ values were calculated [9] |
| Cell Assay |
MG-132 is added to cells at different concentrations for 24, and 48 hours. Centrifugation is used to collect the supernatant and monolayer cells, which are then preserved in 70% ethanol in PBS before being stained with acridine orange. Acridine orange (5 mg/mL in PBS) and equal volumes of cells are combined on a microscope slide, and fluorescence microscopy is used to examine the mixture. Cells are collected by centrifugation and stained with propidium iodide and annexin V for annexin V analysis. Propidium iodide (5 mg/mL) staining is done after rehydrating cells in PBS at room temperature for ten minutes in order to analyze the cell cycle. Utilizing a Coulter Epics XL flow cytometer, every sample is examined. HeLa, A549, and MCF-7 cells were seeded in 96-well plates at 5×10³ cells/well and treated with MG-132(R) (0.05-5 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For cell cycle analysis, cells were treated with 0.2-1 μM drug for 24 hours, stained with propidium iodide, and analyzed by flow cytometry [4] Human bronchial epithelial cells were treated with MG-132(R) (0.1-1 μM) for 1 hour before TNF-α stimulation. After 24 hours, cell lysates were analyzed by Western blot to detect IκBα degradation, and IL-6/IL-8 levels in supernatants were quantified by ELISA [2] SW1353 cells were treated with MG-132(R) (0.2-2 μM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI staining, and protein expression (cleaved caspase-3, Bcl-2) was assessed by Western blot. U251 cells were treated with 0.5-2 μM drug for 24 hours, and E-cadherin expression was detected by immunofluorescence and Western blot [6,8] |
| Animal Protocol |
Male mdx (C57BL/10ScSn DMD mdx) mice ~10 μg/kg/day Injection Nude mice bearing HeLa xenografts (100-150 mm³) were randomly divided into control and treatment groups. MG-132(R) was dissolved in DMSO and diluted with saline (final DMSO concentration ≤ 5%), then administered intraperitoneally at 0.5 mg/kg/day for 14 days. Tumor volume was measured every 2 days, and mice were euthanized to collect tumors for Western blot analysis of ubiquitinated proteins and caspase-3 [4] Rheumatoid arthritis was induced in DBA/1 mice using type II collagen. Mice were treated with MG-132(R) (0.3 mg/kg/day) via intraperitoneal injection for 21 days. Joint swelling was measured weekly, and serum TNF-α/IL-6 levels were quantified by ELISA. Joint tissues were harvested for histopathological analysis [7] Nude mice with A549 xenografts were treated with MG-132(R) intraperitoneally at 0.7 mg/kg/day for 18 days. Survival time was recorded daily, and tumor tissues were processed for immunohistochemical staining of VEGF and CD31 (angiogenesis marker) [6] |
| Toxicity/Toxicokinetics |
In vitro, MG-132(R) showed mild cytotoxicity to normal human fibroblasts (MRC-5) with an IC₅₀ of 5.2 μM, indicating a favorable therapeutic index compared to cancer cells [4] Mice treated with MG-132(R) at 0.5 mg/kg/day (i.p.) for 14 days showed no significant weight loss or organ toxicity. Serum ALT, AST, creatinine, and BUN levels were within normal ranges [4] The plasma protein binding rate of MG-132(R) was ~90% in human plasma as determined by equilibrium dialysis [9] |
| References |
[1]. J Biochem . 1996 Mar;119(3):572-6. [2]. Am J Respir Cell Mol Biol . 1998 Aug;19(2):259-68. [3]. Cell Death Differ . 2001 Mar;8(3):210-8. [4]. Cancer Res . 2006 Jun 15;66(12):6379-86. [5]. J Med ChemCancer Res . 2007 Mar 1;67(5):2247-55. [6]. Br J Cancer . 2008 Nov 18;99(10):1613-22. [7]. Am J Pathol . 2003 Oct;163(4):1663-75. [8]. BMC Musculoskelet Disord . 2011 Aug 15:12:185. [9]. J Med Chem . 2010 Feb 25;53(4):1509-18. |
| Additional Infomation |
=AE41 MG-132(R) is a synthetic peptide aldehyde and reversible proteasome inhibitor that blocks the degradation of ubiquitinated proteins, leading to the accumulation of misfolded proteins and induction of cell cycle arrest and apoptosis in cancer cells [1] It has been widely used as a tool compound in research to study the role of the ubiquitin-proteasome system in various biological processes, including cell proliferation, inflammation, and apoptosis [3] Beyond anticancer activity, MG-132(R) exhibits anti-inflammatory effects by inhibiting NF-κB activation, making it a potential candidate for the treatment of inflammatory diseases such as rheumatoid arthritis [7] The drug's poor aqueous solubility and short half-life limit its clinical application, but it serves as a prototype for the development of more stable proteasome inhibitors (e.g., bortezomib) [9] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (5.26 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.26 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. Solubility in Formulation 3: ≥ 0.83 mg/mL (1.75 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. Solubility in Formulation 4: 4% DMSO+30% PEG 300+20% propylene glycol+ddH2O: 2 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1025 mL | 10.5126 mL | 21.0252 mL | |
| 5 mM | 0.4205 mL | 2.1025 mL | 4.2050 mL | |
| 10 mM | 0.2103 mL | 1.0513 mL | 2.1025 mL |