Proteasome

Proteasome Labeling and In-Gel Detection [1]
The potency of MV151 (6) was determined by measuring proteasomal activity by using fluorogenic substrates. EL-4 lysates were incubated with increasing concentrations of MV151, and the cleavage of the substrates Suc-Leu-Leu-Val-Tyr-AMC (chymotrypsin-like activity), Z-Ala-Ala-Arg-AMC (trypsin-like activity), and Z-Leu-Leu-Glu-βNA (peptidylglutamyl peptide hydrolytic activity) was monitored. At concentrations below 1 μM, MV151 appears to inhibit trypsin-like activity and chymotrypsin-like activity more efficiently than it inhibits PGPH activity (Figure 2A). This might be due to differences in activity between the subunits, to allosteric effects, to minor subunit specificities of the probe, or to nonsaturation kinetics. At concentrations of 1 μM and higher, MV151 completely inhibits all three activities.
Direct in-gel visualization of MV151-labeled proteasome subunits was explored by using a fluorescence scanner. Treatment of purified human 20S proteasome with MV151 showed uniform labeling of the active subunits β1, β2, and β5 (Figure 2B). To determine the sensitivity of the in-gel detection, we directly compared fluorescence readout of the gel (Figure 2B) with detection of proteasome subunits by silver staining of proteins (Figure 2C). The in-gel detection was shown to be very sensitive since as little as 3 ng proteasome was sufficient to detect individual MV151-labeled proteasome subunits; detection with this method is at least three times more sensitive than silver staining.
We next compared the labeling of the constitutive β1, β2, and β5 subunits and the immunoproteasome β1i, β2i, and β5i subunits. For this purpose, we labeled the proteasomes in lysates of the human cervix carcinoma cell line HeLa (expressing constitutive proteasome) and the murine lymphoid cell line EL-4 (expressing both constitutive and immunoproteasome) with increasing concentrations of MV151. All active constitutive and inducible β proteasome subunits were neatly and uniformly labeled by MV151 (Figures 2D and 2E). All subunits were already detectable at a concentration of 10 nM MV151 and reached saturation in fluorescence signal at 1 μM MV151. At higher concentrations of MV151, an increased nonspecific labeling was observed in the high molecular weight region.

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Functional Proteasome Inhibition in Living Cells [1]
We next addressed whether MV151 is able to label proteasome subunits in living cells. EL-4 and HeLa cells were incubated with increasing concentrations of MV151. Specific and sensitive labeling of all proteasome subunits was observed in EL-4 (Figure 4A) and HeLa cells (Figure 4B), although higher concentrations were required than for labeling of subunits in lysates. Labeling of the β1 subunit shows a lower intensity than in lysates, whereas β5 labeling looks more pronounced. This difference in the labeling profile between the proteasome in cell lysates and living cells has been previously reported; however, the reason for this remains unclear. Importantly, incubation of EL-4 and HeLa cells with the inactive control compound MV152 (7), which is almost identical to MV151 but lacks the reactive vinyl sulfone warhead, showed no labeling of the proteasome or any other protein (Figures 4A and 4B).

Monitoring of Proteasome Inhibition in Mice [1]
The results obtained in cell lines prompted us to investigate whether MV151 could be used to label proteasomes in mice. To test the bioavailability of MV151, C57Bl/6 mice were given a single intraperitoneal injection with MV151 (20 μmol/kg body weight) and were sacrificed 24 hr postinjection. Fluorescence microscopic analysis of mouse tissues revealed the capacity of MV151 to penetrate tissues in vivo. The highest Bodipy TMR fluorescence was detected in the liver (Figure 5A) and in the pancreas (Figure 5B). Interestingly, Bodipy TMR fluorescence was higher in the peripheries of the tissues, indicating that the probe might reach the liver most efficiently by diffusion from the peritoneal cavity rather than being distributed by entering the bloodstream.

To examine the effect of administration of the proteasome probe, we took advantage of a recently developed transgenic mouse model for monitoring the ubiquitin-proteasome system, which is based on the ubiquitous expression of the UbG76V-GFP reporter. We have previously shown that administration of the proteasome inhibitors epoxomicin and MG262 results in a substantial accumulation of the UbG76V-GFP reporter in affected tissues. The accumulation was primarily found in the liver and at higher concentrations in other tissues. In the present experiment, the UbG76V-GFP reporter mice were given a single intraperitoneal injection with MV151 (20 μmol/kg body weight). A total of 24 hr postinjection, the mice were sacrificed and several tissues were analyzed by fluorescence microscopy. Cells accumulating UbG76V-GFP were detected in the liver (Figure 5C) and the pancreas (Figure 5E), which also contained the highest Bodipy TMR fluorescence of all of the examined tissues (spleen, intestine, kidney, liver, and pancreas). Importantly, all of the cells that accumulated the UbG76V-GFP reporter contained very high Bodipy TMR fluorescence. The proteasome probe was distributed both in the cytoplasm and nuclei of the cells that accumulated the reporter. Similar to our observations from experiments in cell culture, the affected cells in the mice contained granular accumulations of MV151 in the cytoplasm in close proximity to the nucleus. We verified that accumulation of UbG76V-GFP in the liver and pancreas coincided with proteasomal blockade by MV151. SDS-PAGE followed by in-gel fluorescence analysis of liver (Figure 5D) and pancreas (Figure 5F) homogenates of animals treated with MV151 revealed that the proteasome catalytic subunits were labeled as expected, although higher background labeling compared to in vitro studies was observed. (For Figures 5D and 5F, respectively, the tissues from the images in Figures 5C and 5E were used.) SDS-PAGE followed by in-gel fluorescence analysis of spleen homogenates showed labeling of both constitutive and inducible proteasome catalytic subunits (Figure 5G).

As the final set of experiments, we monitored the biodistribution of MG262 in UbG76V-GFP transgenic mice. We selected the boronic acid MG262 for this purpose because it is both commercially available in purified form and most closely resembles the drug Bortezomib. Animals were injected subcutaneously with either 5 μmol/kg or 10 μmol/kg body weight of the boronic acid MG262 and were sacrificed 24 hr postinjection. Spleen and pancreas were lysed and treated with MV151. SDS-PAGE analysis revealed significant reduction of labeled bands corresponding to the proteasome catalytic subunits when compared with tissue lysates from untreated animals (Figure 6A, pancreas; Figure 6B, spleen). Fluorescence microscope analysis of the same tissues (Figures 6A and 6B) confirmed the concentration-dependent inhibition of the proteasome in MG262-treated UbG76V-GFP mice, as indicated by increased levels of UbG76V-GFP reporter accumulation.

Proteasomal Activity Measurement with Fluorogenic Substrates [1]
Protein lysates from EL-4 (1 mg/ml) were incubated with various concentrations of MV151 (6) for 1 hr at 37°C. For measurement of proteasomal activities, 10 μg labeled lysate was added to 100 μl substrate buffer, containing 20 mM HEPES (pH 8.2), 0.5 mM EDTA, 1% DMSO, 1 mM ATP, and 10 μM Z-Ala-Ala-Arg-AMC (tryptic-like), 60 μM Suc-Leu-Leu-Val-Tyr-AMC (chymotryptic-like), or 60 μM Z-Leu-Leu-Glu-βNA (caspase-like). Fluorescence was measured every minute for 25 min at 37°C by using a Fluostar Optima 96-well plate reader (λex/λem = 355/450 nm for AMC and 320/405 nm for βNA), and the maximum increase in fluorescence per minute was used to calculate specific activities of each sample. Nonspecific hydrolysis was assessed by preincubation with 1 μM epoxomicin for 1 hr at 37°C and was subtracted from each measurement.

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In-Gel Detection of Labeled Proteasome Subunits [1]
Whole-cell lysates were made in lysis buffer containing 50 mM Tris (pH 7.5), 1 mM DTT, 5 mM MgCl2, 250 mM sucrose, 2 mM ATP. Protein concentration was determined by the colorimetric Bradford method. For the labeling reactions, 10 μg total protein lysates was incubated for 1 hr at 37°C with increasing concentrations of MV151 in a total reaction volume of 10 μl. Where indicated, 50 ng purified 20S proteasome was used. For competition studies, cell lysates (10 μg) were exposed to the known inhibitors for 1 hr prior to incubation with MV151 (0.1 μM) for 1 hr at 37°C. For assessment of background labeling, heat-inactivated lysates (10 μg, boiled for 3 min with 1% SDS) were treated with MV151. Reaction mixtures were boiled with Laemmli's buffer containing β-mercapto-ethanol for 3 min and were resolved on 12.5% SDS-PAGE. In-gel visualization was performed in the wet gel slabs directly by using the Cy3/Tamra settings (λex 532, λem 560) on the Typhoon Variable Mode Imager. Labeling profiles in living cells were determined by incubating ∼1 × 106 cells with 1–10 μM MV151 in culture medium at 37°C for 8 hr. Cells were lysed, and in-gel detection was carried out as described above.

Microscopy [1]
Some 0.5 × 106 cells were seeded on 35 mm petri dishes with 14 mm microwell nr 1.5 coverslips and on glass-bottomed microwell dishes and were allowed to attach overnight. Cells were visualized with a 60× oil immersion lens on a Nikon Eclipse TE 2000U microscope equipped with a Radiance 2100 MP integrated laser and detection system and a Tsunami Multiphoton laser module. LaserSharp 2K software was used for microscope control and data acquisition, and Image Pro 3DS 5.1 software was used for image processing. GFP was excited at λex = 488 nm and was detected at 500–530 nm. MV151 and MV152 were excited at λex = 543 nm and were detected at 560–620 nm. CLSM images were adjusted for brightness and contrast by using Photoshop software.

Mouse Experiments [1]
Mice were housed according to Swedish animal care protocols with a 12 hr day/light cycle, and they were fed standard laboratory chow and tap water ad libitum. Adult C57Bl/6 and UbG76V-GFP/1 mice [16], matched for sex and age, were given a single intraperitoneal injection of vehicle (60% DMSO, 40% PBS), MV151 (20 μmol/kg body weight), or MG262 (5 or 10 μmol/kg body weight) in a total volume of 200 μl. Based on prior experience in our lab, the boronic acid inhibitors proved to be more potent and showed better tissue penetration in vivo compared to the vinyl sulfone inhibitors. Therefore, the 20 μmol/kg bodyweight dose was chosen for MV151, which showed no apparent toxicity in mice. Mice were euthanized 24 hr postinjection by anesthetization with inhaled isoflurane (4.4% in oxygen), followed by transcardial perfusion with 50 ml PBS for removal of contaminating blood. Tissues collected for immunocytochemical analysis were processed as described previously. Briefly, 12 μm cryosections were fixed for 15 min in 4% paraformaldehyde/PBS and washed in PBS; where mentioned, Hoechst nuclear stain (2 μg/ml in H2O) was applied for 15 min in the dark, followed by washing in PBS. Sections were mounted in a matrix containing 2.5% DABCO. Confocal microscopy was performed on a Zeiss LSM 510 META system. Tissues isolated for in-gel analysis were lysed with a Heidolph tissue homogenizer in 300 μl lysis buffer and were further treated as described above.

[1]. A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo. Chem Biol. 2006 Nov;13(11):1217-26.

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. [1]

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In summary, we described the synthesis of MV151 and characterized it as being a cell-permeable, broad-spectrum proteasome inhibitor. MV151 enables broad-spectrum proteasome profiling, both in cell lysates and in living cells. The Bodipy TMR dye proved to be very useful for in-gel readout of labeled active subunits in that it provided a straightforward method for direct and sensitive proteasome profiling and omitted the need for western blotting, radioactivity, and gel drying. MV151 could be readily detected upon administration to mice and correlated with inhibition of the proteasome in the affected tissues. Finally, MV151-mediated proteasome labeling in combination with UbG76V-GFP transgenic mice is a useful strategy for monitoring the biodistribution of proteasome inhibitors.[1]
The proteasome is a key enzyme in the maintenance of cellular homeostasis. Here, the synthesis and characterization of the activity-based, fluorescent, and cell-permeable MV151 are presented. MV151 targets the proteasome specifically and shows broad-spectrum activity by covalent and irreversible binding to the catalytic N-terminal threonine residues of immunoinducible and constitutively active β subunits. The bright fluorophore facilitates rapid and sensitive detection of active proteasome subunits by in-gel detection and fluorescence microscopy in living cells. Potentially, MV151 can find application in diverse fields of proteasome research: in medical research, for profiling the active proteasome fractions in a clinically relevant sample; and in chemistry and biochemistry, facilitating rapid determination of potency and subunit specificity of new proteasome inhibitors. [1]

C59H91BN8O9F2S

1137.27

1136.669

C, 62.31; H, 8.07; B, 0.95; F, 3.34; N, 9.85; O, 12.66; S, 2.82

945611-88-1

945611-88-1 (MV-151); 945611-89-2 (MV-152)

154732122

Typically exists as solid at room temperature

6

12

36

80

2280

3

[B-]1(N2C(=C(C(=C2C=C3[N+]1=C(C=C3)C4=CC=C(C=C4)OC)C)CCC(=O)NCCCCCC(=O)NCCCCCC(=O)NCCCCCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)/C=C/S(=O)(=O)C)C)(F)F

JNIDQOOVQFMCNR-FWDZYPIVSA-N

InChI=1S/C59H91BF2N8O9S/c1-40(2)36-46(31-35-80(10,77)78)66-58(75)51(38-42(5)6)68-59(76)50(37-41(3)4)67-57(74)22-16-13-19-33-64-55(72)20-14-11-17-32-63-54(71)21-15-12-18-34-65-56(73)30-28-49-43(7)53-39-47-25-29-52(45-23-26-48(79-9)27-24-45)70(47)60(61,62)69(53)44(49)8/h23-27,29,31,35,39-42,46,50-51H,11-22,28,30,32-34,36-38H2,1-10H3,(H,63,71)(H,64,72)(H,65,73)(H,66,75)(H,67,74)(H,68,76)/b35-31+/t46-,50+,51+/m1/s1

(2S)-2-[6-[6-[6-[3-[2,2-difluoro-12-(4-methoxyphenyl)-4,6-dimethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaen-5-yl]propanoylamino]hexanoylamino]hexanoylamino]hexanoylamino]-4-methyl-N-[(2S)-4-methyl-1-[[(E,3S)-5-methyl-1-methylsulfonylhex-1-en-3-yl]amino]-1-oxopentan-2-yl]pentanamide

MV151; MV 151; 945611-88-1; (2S)-2-[6-[6-[6-[3-[2,2-difluoro-12-(4-methoxyphenyl)-4,6-dimethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaen-5-yl]propanoylamino]hexanoylamino]hexanoylamino]hexanoylamino]-4-methyl-N-[(2S)-4-methyl-1-[[(E,3S)-5-methyl-1-methylsulfonylhex-1-en-3-yl]amino]-1-oxopentan-2-yl]pentanamide; 6-(3-(5,5-difluoro-7-(4-methoxyphenyl)-1,3-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-2-yl)propanamido)-N-((4S,7S,10S)-7,10-diisobutyl-2-methyl-4-((E)-2-(methylsulfonyl)vinyl)-6,9,12,19-tetraoxo-5,8,11,18-tetraazatetracosan-24-yl)hexanamide; MV-151; Bodipy TMRAhx(3)L(3)VS.

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.)