Proteasome

The mechanisms involved in the anti-angiogenic actions of the proteasome inhibitors are poorly understood. Here, we report that the gene expression of the VEGF receptor Flt-1 (vascular endothelial growth factor receptor 1) was down-regulated by the reversible proteasome inhibitor MG-262 in explant cultures of the developing chicken pecten oculi, a vascular organ consisting of endothelial cells, pericytes, and macrophages. In addition, the inhibitor prevented the induction of Flt-1 by lipopolysaccharide (LPS) in macrophages and down-regulated the expression of Flt-1 after LPS induction. Flt-1 gene expression was also down regulated by MG262 in cultures of human microvascular endothelial cells. Interestingly, a transcript of Flt-1, coding for a soluble form of the receptor (sFlt-1) with anti-angiogenic properties, was not down-regulated in the same extent. Only a small decrease in the expression of VEGF and Ang-2 was detected in the pecten oculi upon inhibition of the proteasome, while no major changes were observed in the expression of other angiogenic molecules, such as KDR or Ang-1. Since recent experiments have demonstrated the importance of anti-Flt-1 therapy in the inhibition of tumor angiogenesis, retinal angiogenesis, arthritis, and atherosclerosis (Luttun et al. [2002]: Nat Med 8:831-840), our observation on down-regulation of Flt-1 in microvascular endothelial cells and macrophages by MG262 supports the postulated role of the proteasome inhibitors as potential candidates for therapeutic modulation of angiogenesis and inflammation.[1]

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Proteasome inhibitors increased IL-8 and AP-1 luciferase activity [2]
To determine the gene regulatory effect of proteasome inhibitors on IL-8 expression, we performed reporter gene assays to test four proteasome inhibitors, MG132, aLLN, lactacystin, and MG262. As shown in Fig. 1 , these four proteasome inhibitors increased IL-8 promoter activity in a concentration-dependent manner. MG262 was the most potent proteasome inhibitor to achieve IL-8 stimulation within 0.1 nM–1 μM. MG132 and lactacystin, on the other hand, exhibited a moderate and comparable potency within 0.1–50 μM. Among the proteasome inhibitors tested, aLLN was the weakest one to stimulate IL-8 response. The stimulation efficacy at the highest concentration of these inhibitors examined, 1 μM for MG-262, 50 μM for MG132 and lactacystin, was around a 12–14-fold increase, while that for 50 μM of aLLN was about eight-fold. At these concentrations examined, no cytotoxicity was seen. However, increasing the concentrations of MG132, lactacystin and aLLN to 100 μM resulted in cytotoxicity within 24 h, as assessed from the MTT assay (data not shown).

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Proteasome inhibitors inhibited cytokine-induced κB reporter activity [2]
To verify the inhibitory action of these proteasome inhibitors on NF-κB transcription, the κB-luciferase reporter gene assay was carried out. The results revealed that while κB luciferase activity in basal condition is not altered by MG132, aLLN, lactacystin, or MG-262, its stimulation by two potent NF-κB inducing cytokines, IL-1β (10 ng/ml) and tumor necrosis factor α (TNF-α) (50 ng/ml), was antagonized by the presence of proteasome inhibitors (data not shown). The IC50 values against cytokine-induced NF-κB activation were 0.1–0.3 μM for MG132, 1 μM for aLLN and lactacystin, and 1–3 nM for MG262.

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ROS-dependent effects of proteasome inhibitors [2]
Since several reports have indicated AP-1 transcription factor as a target of ROS , we next wanted to address the role of ROS in the action of proteasome inhibitors. Another reason to address this issue is the recent finding that lactacystin can lead to significant oxidative damage in NT-2 and SK-N-MC cell lines. Fig. 3 shows that when the antioxidant NAC (3 or 10 mM) or GSH (5 or 30 mM) was pretreated for 15 min, the increased extents of IL-8 and AP-1 reporter activities caused by proteasome inhibitors were markedly attenuated. This result suggested the involvement of ROS as intermediators in the signaling pathway induced by proteasome inhibitors. To verify this suggestion, we used the fluorescent agent DCFH-DA to measure the intracellular content of ROS. When examining the concentrations of these proteasome inhibitors that induced maximal and comparable stimulation on IL-8 and AP-1 (10 μM for MG132, aLLN and lactacystin, and 0.1 μM for MG-262), the results shown in Fig. 4 indicated the time-dependent increases of intracellular ROS following exposure to proteasome inhibitors. Within 3 h incubation, the effects of MG132, lactacystin and MG-262 on ROS production exhibited biphasic features, which peaked at 5 min, declined gradually and re-induced around 1 h. The efficacy in terms of the rapid stimulation at 5 min is MG262>lactacystin>MG132, aLLN, while that occurring at 1 h is MG132>MG262, lactacystin>aLLN. When examining MG132, aLLN and lactacystin at 0.1 and 1 μM, and MG262 at 0.01 μM, the ROS production at 5 min was also increased by 40–80% (data not shown).

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GA augments proteasome inhibitor-induced inhibition of cell viability in malignant cells [3]
To verify our hypothesis and determine whether GA can sensitize cancer cells to treatment with proteasome inhibitors, we first tested the effects of various concentrations of gambogic acid alone on the viability of human leukemia K562 and murine hepatocarcinoma H22 cells. The results showed that even at the maximal dose tested, GA as a single agent inhibited cell viability by less than 20% in both cell lines (Fig. 1B and C). However, when the two cell lines were treated with the proteasome inhibitor MG262 at different doses with or without 0.4 µM of GA for 48 h, inhibition of cell viability was dramatically increased, for example, from 5% when treated with 6.25 nM of MG-262 alone to 82% when combined with GA (K562 cells, Fig. 1D). When H22 cells were treated under the same treatment conditions, the addition of GA boosted the inhibitory effects of MG262 on cell viability/proliferation from 11% to 73% (Fig. 1E). GA was also tested in a combinatorial manner with another proteasome inhibitor MG132 in both K562 and H22 cell lines, and a similar synergistic effect was observed (Fig. 1F and G). An analysis of the combination index (CI) indicated that all values of CI were less than 1 (CI < 1), indicating that GA and proteasome inhibitors possess synergistic effects on inhibition of cancer cell viability (Fig. 1H and I).

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GA is able to sensitize malignant cells to proteasome inhibitor-induced apoptotic cell death [3]
We and others have reported that inhibition of proteasome activity, particularly inhibition of chymotrypsin-like activity, is associated with induction of apoptosis in malignant cells. To determine whether GA has synergistic effects on proteasome inhibitor-induced apoptosis, H22 and K562 cells were treated with MG-262 or MG132 in the presence or absence of GA, followed by the Annexin V Apoptosis assay for flow cytometric analysis. The results showed that co-treatment of these malignant cells, especially K562 cells, dramatically increased the population of both Annexin V and propidium iodide (PI) stained cells (Fig. 2A and B), indicating that the combination treatment induces more cell death than each treatment alone.

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Potential mechanisms of synergistic effects of GA on proteasome inhibitor-induced apoptosis and antitumor activity [3]
The results from our in vitro and in vivo studies have demonstrated that the combination treatment of GA and proteasome inhibitors results in synergistic inhibition of cell growth in malignant cultures and tumors grown in animal models. To explore potential mechanisms involved, we treated K562 cells with 0.4 µM GA, 25 nM MG262, various concentrations of MG132 as single agents, or in their combinations (GA + MG132 or GA + MG-262) for 12 or 24 h, followed by Western analysis to measure caspase activation and PARP cleavage. After 12-h treatment, the GA + either proteasome inhibitor, but not GA or proteasome inhibitor alone, could induce cleavage of PARP as well as cleavage/activation of caspases 8 and 9 (Fig. 4A, left panel). Similar results were observed in the cells treated for 24 h (Fig. 4A, right panel). These findings indicate that GA is able to enhance the proteasome-inhibitor-induced apoptosis via caspase-dependent pathways.

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Monitoring the ubiquitin/proteasome system in primary cells [5]
To study the functionality of the UbG76V-GFP transgene, we established primary cell cultures from different tissues and treated them with different classes of proteasome inhibitors: (i) the reversible peptide aldehyde inhibitor MG-132 (ref. 34, ii) the reversible peptide boronate inhibitor MG-262 (ref. 35, iii) the irreversible peptide vinylsulfone inhibitor Z-L3-VS (ref. 36) and (iv) the irreversible natural compound inhibitor epoxomicin20. Although the peptide boronates and peptide aldehydes are both reversible inhibitors, the peptide boronates are more potent owing to their slower dissociation rates.
Primary fibroblasts, cardiomyocytes and neurons of UbG76V-GFP mice responded to treatment with 10 μM MG-132, 0.5 μM MG-262, 10 μM Z-L3-VS or 0.5 μM epoxomicin with pronounced accumulation of the UbG76V-GFP reporter as visualized by fluorescence microscopy (Fig. 2a and data not shown). Accumulation of the UbG76V-GFP was evident throughout the cytoplasm and nucleus, with a slightly stronger signal in the latter. GFP fluorescence emission, as quantified by flow cytometry, showed an approximately tenfold increase in primary fibroblasts treated with 0.5 μM epoxomicin as compared to untreated controls (Fig. 2b). A modest increase in GFP fluorescence could already be observed after 5 h of treatment with epoxomicin and further increased up to tenfold after 15 h of treatment (Fig. 2c). Titration of the MG-132, MG-262 and epoxomicin inhibitors demonstrated that the accumulation of UbG76V-GFP was dose dependent (Fig. 2d).

Monitoring the ubiquitin/proteasome system in vivo [5]
To evaluate the functionality of the reporter in vivo, we administered intraperitoneally the inhibitors MG-132 (10 μmol/kg body weight), epoxomicin or MG-262 (both 1 μmol/kg body weight) or vehicle only (60% dimethyl sulfoxide (DMSO)) to UbG76V-GFP/1 mice. On the basis of the kinetics of fluorescence accumulation observed in primary cultures and in a stable transfectant31,38, the animals were killed after 20 h, and liver, lung, brain, pancreas, kidney, spleen and small intestine were examined by fluorescence microscopy. Fluorescent cells were detected exclusively in the livers of the mice treated with epoxomicin and MG-262, whereas no fluorescent cells were detected in mice treated with MG-132 and vehicle only (Fig. 3a). Similar levels of fluorescence were detected in the livers of animals treated with epoxomicin-and MG-262, but the distribution of fluorescent cells was markedly different. Treatment with MG-262 resulted in accumulation of GFP in the vast majority of hepatocytes distributed throughout the liver, whereas in epoxomicin-treated animals the fluorescent cells were primarily localized along the margins of the liver and in small, defined areas inside the liver lobes. The centrally located positive cells were positioned around the liver triads, which contain the portal vein, whereas the area surrounding the central vein remained negative for GFP (Fig. 3b). The more pronounced accumulation of UbG76V-GFP in cells surrounding the portal veins was also confirmed by immunohistochemistry using GFP-specific antibodies (Fig. 3c). This highly sensitive immunostaining confirmed the complete absence of the UbG76V-GFP in hepatocytes surrounding the liver triads of transgenic mice treated with vehicle only (Fig. 3d).

Analysis of the chymotrypsin-like activity of the proteasomes in liver lysates of mice treated with the different inhibitors confirmed the relative potency of the compounds' action on the proteasomes as indicated by the UbG76V-GFP fluorescence: strong inhibition was found in mice after MG-262 injection, modest inhibition after epoxomicin injection and no inhibition after MG-132 injection (Fig. 4). Proteasomal activities in kidney and spleen lysates were inhibited to a similar extent, though more activity was present in these tissues than in the liver, confirming that the liver is the primary effector site after intraperitoneal administration of proteasome inhibitors.

In the next set of experiments, UbG76V-GFP/1 mice were injected with 1 or 5 μmol/kg body weight MG-262. A clear dose-dependent increase in GFP fluorescence was observed in the liver (Fig. 5a,b). Moreover, although low doses of the inhibitor did not cause GFP accumulation in the small intestine (Fig. 5c), bright fluorescence was detected after treatment with the high dose (Fig. 5d). A similar dose-dependent accumulation of UbG76V-GFP was also observed in the pancreas (data not shown and Fig. 5e, 5 μmol/kg) and kidney (data not shown and Fig. 5f, 5 μmol/kg), whereas only a small fraction of the cells became fluorescent at the higher concentration in the lung (Fig. 5g) and spleen (Fig. 5h). No fluorescent cells could be detected in the brain, heart and skeletal muscles. A dose-dependent decrease of the chymotrypsin-like activity of the proteasome was observed in lysates of the liver, kidney and spleen from MG-262–treated animals (Fig. 4). Intriguingly, very similar inhibition levels were observed in the kidney and spleen from MG-262-treated animals even though these organs showed different levels of functional impairment of the ubiquitin/proteasome system, with only a few affected cells in the spleen as compared to a systemic accumulation of the reporter in the kidney. Notably, clear signs of toxicity, including hypothermia, unresponsiveness and weight loss, were detected upon treatment with the high MG-262 dose, whereas the low dose had no apparent effect.

Transfection and reporter gene assay [2]
For transfection assays, 5×105 HEK293 cells were seeded into six-well plates. Cells were transfected on the following day by the calcium phosphate precipitation method. Premix DNA with 33.4 μl 0.1×TE buffer, 12.6 μl 1 M CaCl2 in a tube for each well, then mix slowly with 46 μl 2×Hanks’ balanced salt solution in 25 s. Incubate the mixture for 25 min at room temperature, and add into each well. After 24 h incubation, transfection was complete, and cells were incubated with the indicated concentrations of proteasome inhibitors. After another 24 h incubation, the media were removed, and the cells were washed once with cold phosphate-buffered saline. To prepare lysates, 100 μl of reporter lysis buffer was added to each well, cells were scraped from dishes. Collect the supernatant after centrifugation at 13 000 rpm for 30 s. Aliquots of cell lysates (5 μl) containing equal amounts of protein (10–20 μg) were placed into the wells of an opaque black 96-well microplate. An equal volume of luciferase substrate was added to all samples, and the luminescence was measured in a microplate luminometer. The luciferase activity value was normalized to transfection efficiency monitored by the cotransfected β-galactosidase expression vector, and is presented as the percentage of luciferase activity measured in the presence of each proteasome inhibitor relative to the activity of control cells with no stimulation.

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Flow cytometry for ROS formation [2]
2′,7′-Dichlorodihydrofluorescin diacetate (DCFH-DA) was used as an indicator for the formation of intracellular reactive oxygen species (ROS). Cells were pretreated with DCFH-DA (50 μM) for 30 min, and then indicated concentrations of proteasome inhibitors were added for different time periods. Once ROS was generated, the DCFH oxidation product, DCF fluorescence can be detected by flow cytometer. The fluorescence was assessed by counts of FL1-H, and the mean value would represent the ability of a chemical compound to induce ROS formation.

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MTS assay [3]
The effects of compounds on cell viability were determined by the MTS assay. The MTS tetrazolium compound is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium. This conversion is presumably accomplished by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. Exponentially growing cells were harvested and seeded at 2500 cells/well in a 96-well plate. After 6 h incubation, compounds or DMSO, as the untreated control, were added, followed by continuous incubation for 48 h. Twenty microliter of MTS were added to each well and the incubation was continued for an additional 3 h. The absorbance was measured with a microplate reader at 490 nm. The percent inhibition was calculated as follows: Inhibition rate (IR) (%) = [1 – (absorbance of experimental well – absorbance of blank)/(absorbance of untreated control well – absorbance of blank)] × 100%.

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Cell death detection assay via flow cytometry [3]
Apoptotic cell death was measured by Annexin V-FITC and propidium iodide (PI) double staining followed by flow cytometry as previously described. Briefly, cultured K562 and H22 cells were harvested and washed with cold PBS and resuspended with the binding buffer, followed by Annexin V- FITC incubation for 15 min and PI staining for another 15 min at 4°C in dark. The stained cells were analyzed with flow cytometry within 30 min.

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Morphological characterization of cell death [3]
K562 or H22 cells were treated as described. To monitor temporal changes in the incidence of cell death in the live culture condition, propidium idodide (PI) was added to the cell culture medium and at the desired sequential time points, the cells in the culture dish were imaged with an inverted fluorescence microscope equipped with a digital camera. PI is not able to enter the normal live cells but the dying or dead cells lose their membrane integrity and PI can enter their nucleus, bind to double-stranded DNA, and thereby positively stain the dying and dead cells.

Fluorescence analysis and immunohistochemical staining of cryosections.[5]
Adult transgenic mice were treated by intraperitoneal injection of 200 μl of vehicle only (60% DMSO) or epoxomicin (Affinity), MG-132 (carboxybenzyl-leucyl-leucyl-leucinal), MG-262 and Z-L3-VS (carboxybenzyl-leucyl-leucyl-leucine vinyl sulphone). Tissues were excised 20 h after injection, fixed overnight in 4% paraformaldehyde and immersed in a graded sucrose series. For immunohistochemical analysis, 10 μm cryosections were fixed with 4% paraformaldehyde and treated for 30 min with 3% H2O2. The sections were blocked in goat serum and incubated with polyclonal anti-GFP antibody at 4 °C overnight. After washing, biotinylated anti–rabbit IgG secondary antibody was added for 30 min at 20 °C, and then the sections were incubated with avidin-biotin-peroxidase complex for 30 min and stained with diaminobenzidine.

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Measurement of proteasomal activity.[5]
Mice were injected intraperitoneally with the indicated amount of proteasome inhibitors or DMSO only and killed after 20 h. Liver, kidney and spleen were homogenized in buffer containing 50 mM Tris pH 7.5, 5 mM MgCl2, 1 mM dithiothreitol and 250 mM sucrose and centrifuged for 10 min at 14,000g at 4 °C. The chymotryspin-like activity in the lysates was determined by fluorimetric quantification of the hydrolysis of the fluorogenic substrate suc-LLVY-AMC as described previously31. Nonproteasomal chymotrypsin-like activity was determined by measuring suc-LLVY-AMC hydrolysis in the presence of 1 μM MG-262.

[1]. Down-regulation of Flt-1 gene expression by the proteasome inhibitor MG262. J Cell Biochem. 2003 Aug 15;89(6):1138-47.

[2]. Proteasome inhibitors stimulate activator protein-1 pathway via reactive oxygen species production. FEBS Lett. 2002 Aug 28;526(1-3):101-5.

[3]. Gambogic acid enhances proteasome inhibitor-induced anticancer activity. Cancer Lett. 2011 Feb 28;301(2):221-8.

[4]. A novel transgenic mouse model reveals deregulation of the ubiquitin-proteasome system in the heart by doxorubicin. FASEB J. 2005 Dec;19(14):2051-3.

[5]. A transgenic mouse model of the ubiquitin/proteasome system. Nat Biotechnol. 2003 Aug;21(8):897-902.

In this report we explored the effects of proteasome inhibitors (MG132, aLLN, lactacystin and MG-262) on interleukin-8 (IL-8) induction. In HEK293 cells, proteasome inhibitors could concentration-dependently increase IL-8 promoter and activator protein-1 (AP-1) activities, but inhibited nuclear factor (NF)-kappa B activation induced by cytokines. The stimulating effects on IL-8 promoter and AP-1 were reduced by N-acetylcysteine, glutathione, diphenyleneiodonium, rotenone and antimycin A. Fluorescent analysis using 2',7'-dichlorodihydrofluorescin diacetate further confirmed the abilities of proteasome inhibitors to induce reactive oxygen species (ROS) production. These results suggest that ROS production by proteasome inhibitors leads to AP-1 activation, which in the absence of NF-kappa B activation still transactivates IL-8 gene expression.[2]

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Proteasome inhibition has emerged as a novel approach to anticancer therapy. Numerous natural compounds, such as gambogic acid, have been tested in vitro and in vivo as anticancer agents for cancer prevention and therapy. However, whether gambogic acid has chemosensitizing properties when combined with proteasome inhibitors in the treatment of malignant cells is still unknown. In an effort to investigate this effect, human leukemia K562 cells, mouse hepatocarcinoma H22 cells and H22 cell allografts were treated with gambogic acid, a proteasome inhibitor (MG132 or MG-262) or the combination of both, followed by measurement of cellular viability, apoptosis induction and tumor growth inhibition. We report, for the first time, that: (i) the combination of natural product gambogic acid and the proteasome inhibitor MG132 or MG-262 results in a synergistic inhibitory effect on growth of malignant cells and tumors in allograft animal models and (ii) there was no apparent systemic toxicity observed in the animals treated with the combination. Therefore, the findings presented in this study demonstrate that natural product gambogic acid is a valuable candidate to be used in combination with proteasome inhibitors, thus representing a compelling anticancer strategy. [3]

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Ubiquitin-proteasome system (UPS) mediated proteolysis is responsible for the degradation of majority of cellular proteins, thereby playing essential roles in maintaining cellular homeostasis and regulating a number of cellular functions. UPS dysfunction was implicated in the pathogenesis of numerous disorders, including neurodegenerative disease, muscular dystrophy, and a subset of cardiomyopathies. However, monitoring in vivo functional changes of the UPS remains a challenge, which hinders the elucidation of UPS pathophysiology. We have recently created a novel transgenic mouse model that ubiquitously expresses a surrogate protein substrate for the UPS. The present study validates its suitability to monitor in vivo changes of UPS proteolytic function in virtually all major organs. Primary culture of cells derived from the adult transgenic mice was also developed and tested for their applications in probing UPS involvement in pathogenesis. Applying these newly established in vivo and in vitro approaches, we have proven in the present study that doxorubicin enhances UPS function in the heart and in cultured cardiomyocytes, suggesting that UPS hyper-function may play an important role in the acute cardiotoxicity of doxorubicin therapy. [4]

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Impairment of the ubiquitin/proteasome system has been proposed to play a role in neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although recent studies confirmed that some disease-related proteins block proteasomal degradation, and despite the existence of excellent animal models of both diseases, in vivo data about the system are lacking. We have developed a model for in vivo analysis of the ubiquitin/proteasome system by generating mouse strains transgenic for a green fluorescent protein (GFP) reporter carrying a constitutively active degradation signal. Administration of proteasome inhibitors to the transgenic animals resulted in a substantial accumulation of GFP in multiple tissues, confirming the in vivo functionality of the reporter. Moreover, accumulation of the reporter was induced in primary neurons by UBB+1, an aberrant ubiquitin found in Alzheimer disease. These transgenic animals provide a tool for monitoring the status of the ubiquitin/proteasome system in physiologic or pathologic conditions.[5]

C25H42BN3O6

491.43

491.317

C, 61.10; H, 8.61; B, 2.20; N, 8.55; O, 19.53

179324-22-2

490002

White to off-white solid powder

4.706

5

6

15

35

653

3

B([C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)OCC1=CC=CC=C1)(O)O

MWKOOGAFELWOCD-FKBYEOEOSA-N

InChI=1S/C25H42BN3O6/c1-16(2)12-20(24(31)29-22(26(33)34)14-18(5)6)27-23(30)21(13-17(3)4)28-25(32)35-15-19-10-8-7-9-11-19/h7-11,16-18,20-22,33-34H,12-15H2,1-6H3,(H,27,30)(H,28,32)(H,29,31)/t20-,21-,22-/m0/s1

[(1R)-3-methyl-1-[[(2S)-4-methyl-2-[[(2S)-4-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]pentanoyl]amino]butyl]boronic acid

MG 262; MG-262; PS-III; 179324-22-2; Z-Leu-Leu-Leu-B(OH)2; CHEMBL114388; [(1R)-3-methyl-1-[[(2S)-4-methyl-2-[[(2S)-4-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]pentanoyl]amino]butyl]boronic acid; 549V4DP94W; MG 262

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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