Calpain

Protease Activity Assays [1]
Peptides were evaluated for ability to bind and subsequently inhibit the cysteine proteases using standard proteolytic fluorescence activity assays. Inhibition was assayed using a standard donor-quencher strategy using a previously published peptide substrates. Enzyme concentration for Calpain-1 was 25 nM. Enzyme concentration for papain was 25 nM. Enzyme concentrations for cathepsin B and cathepsin L was 3 nM. Calpain and papain buffer contained 10 mM dithioreitol (DTT), 100 mM KCl, 2 mM EGTA, 50 mM Tris-HCl (pH 7.5), and 0.015% Brij-35. Substrate concentration for calpain and papain was 0.25 µM H-Glu(Edans)-Pro-Leu-Phe- Ala-Glu-Arg-Lys(Dabcyl)-OH (Km calculation in Supporting Information, Figures S8 & S10). Cathepsin buffer contained 10 mM DTT, 500 mM sodium acetate (pH 5.5), and 4 mM EGTA. Substrate concentration for the cathepsins was 0.25 µM Z-FR-Amc. Calpain was activated by the injection of CaCl2 to a final concentration of 5 mM. Papain and cathepsin assays were activated by the addition of the substrate via a multichannel pipette. Varying concentrations of inhibitor, 1–100 µM, were used for each assay. All assays were done at a total well volume of 100 µL in 96-well plate, and each well contained a separate inhibitor concentration. Fluorescence was read in a Berthold Tri-Star fluorimeter. The excitation wavelength was 380 nm and the emission wavelength was 500 nm for H-Glu(Edans)-Pro-Leu-Phe-Ala-Glu-Arg-Lys(Dabcyl)-OH. The excitation wavelength 351 nm and emission wavelength was 430 nm for Z-FR-Amc.

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Kinetic analysis of Calpain-1 by 3c [1]
To identify inhibition type we used standard Michaelis-Menten treatment. Initial velocities (obtained from the linear segment of the progress curves) were plotted against substrate concentration. Due to the linearity of the first segment of the progress curve we believe that autoproteolysis during the first 500 seconds was not substantial enough to prevent the use of simple Michaelis-Menten kinetics, i.e. loss of enzyme did not change the velocity enough to cause it to deviate from linearity and incorporation of this additional complex would severely complicate the kinetics. Velocities were determined in RFU/sec then converted to µM/sec using the conversion factor 1386 RFU/µM. The conversion factor was obtained by the total hydrolysis of the substrate H-Glu(Edans)-Pro-Leu-Phe-Ala-Glu-Arg-Lys(Dabcyl)- OH in a known concentration by papain. To avoid weighting errors we used the values of Kmapp and Vmaxapp determined directly from the non-linear least-squares best fits of the untransformed data and put these values into the reciprocal equation:

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Determination of IC50 against Enzymes [1]
IC50 curves were generated identifying the initial rate of the enzyme at each inhibitor concentration from the respective progress curves. The conversion factor (1386 RFU/µM) was obtained by the total hydrolysis of the substrate H-E(Edans)-PLFAER-K(Dabcyl)-OH in a known concentration by papain. Initial velocities were converted from RFU/sec to µM/sec. Fractional activity was calculated by dividing the initial velocity at each inhibitor concentration by the initial velocity of the uninhibited enzyme. Data obtained up to 500 seconds was used for the initial rate calculation. The initial rate was then plotted against the log of the inhibitor concentration, and IC50 was calculated by GraphPad Prism.

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Activity Based Probe Linker Experiments [1]
Experimental conditions included 10 mM dithioreitol (DTT), 1.5 µg calpain, 100 mM KCl, 2 mM EGTA, 50 mM Tris-HCl (pH 7.5), 0.015% Brij-35, and either 1 µM or 10 µM of biotinylated probe (DCG-04, NM-01, NM-02, NM-03). Calpain was activated by the addition of calcium (3.33 µM of 50 mM CaCl2) to a final concentration of 8.3 mM in tubes containing either 1 µM or 10 µM ABP. For the negative control, water, instead of CaCl2, was added to the calpain solution containing 10 µM probe. Probes were allowed to bind to the calpain for 20 minutes at room temperature. The reaction was stopped by the addition of 10 µL NuPage® LDS Running Buffer (Life Technologies, Grand Island, NY). 10 µL of each labeled enzyme was loaded on a 10% Bis-Tris NuPAGE® gel and separated via gel electrophoresis for 1.5 h, 140 V. The bands were then transferred to a PVDF membrane at 30 V for 70 min. The membrane was blocked and blotted using the Vectastain® Elite® ABC kit. Kodak film was exposed to the membrane and developed.

[1]. Development of α-helical calpain probes by mimicking a natural protein-protein interaction. J Am Chem Soc. 2012 Oct 24;134(42):17704-13.

We designed a highly specific calpain inhibitor by mimicking the native protein-protein interaction between calpain and its endogenous inhibitor, calpain repressor protein. To achieve this, we developed a novel method to stabilize the α-helix structure in small peptides. This method involved screening 24 commercially available cross-linking agents to identify those capable of successfully alkylating cysteine residues in model peptide sequences. We investigated the effect of cross-linking on the α-helix structure of the selected peptides using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, revealing that cross-linking agents with higher structural rigidity were most effective in stabilizing the α-helix structure. We applied this strategy to the design of calpain inhibitors based on calpain repressor proteins (intrinsically unstable peptides that form specific structures upon binding to enzymes). We stabilized a double-loop α-helix structure that binds to the proximal end of the active site cleft, resulting in a highly efficient and selective calpain inhibitor. We further expanded the application of this inhibitor by developing irreversible calpain family active probes (ABPs) that retain the specificity of the stabilized helix inhibitor. We believe that this inhibitor and ABPs will contribute to future research on calpain, while cross-linking technology will aid in exploring other protein-protein interactions. [1]

C72H97N17O16S

1488.71

1487.702

1914987-47-5

131698206

{Glu(EDANS)}-Pro-Leu-Phe-Ala-Glu-Arg-{Lys(DABCYL)}

EPLFAERX; {Glu(EDANS)}-PLFAER-{Lys(DABCYL)}

Typically exists as solid at room temperature

1.4±0.1 g/cm3

1.658

4.59

15

23

43

106

3080

8

S(C1C=CC=C2C=1C=CC=C2NCCN[C@H](C(N1CCC[C@H]1C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(O)=O)CCCCNC(=O)C1C=CC(N=NC2C=CC(N(C)C)=CC=2)=CC=1)=O)CCCNC(=N)N)=O)CCC(=O)O)=O)C)=O)CC1C=CC=CC=1)=O)CC(C)C)=O)=O)CCC(=O)N)(O)(=O)=O

ZBTCIQPDYVIPOU-DAPRKCRDSA-N

InChI=1S/C72H97N17O16S/c1-43(2)41-57(84-69(100)59-22-14-40-89(59)70(101)55(33-35-62(92)93)87-106(104,105)60-23-12-17-50-51(60)18-11-20-52(50)76-39-36-73)68(99)83-58(42-45-15-7-6-8-16-45)67(98)79-44(3)63(94)80-54(32-34-61(90)91)66(97)81-53(21-13-38-78-72(74)75)65(96)82-56(71(102)103)19-9-10-37-77-64(95)46-24-26-47(27-25-46)85-86-48-28-30-49(31-29-48)88(4)5/h6-8,11-12,15-18,20,23-31,43-44,53-59,76,87H,9-10,13-14,19,21-22,32-42,73H2,1-5H3,(H,77,95)(H,79,98)(H,80,94)(H,81,97)(H,82,96)(H,83,99)(H,84,100)(H,90,91)(H,92,93)(H,102,103)(H4,74,75,78)/t44-,53-,54-,55-,56-,57-,58-,59-/m0/s1

(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[5-(2-aminoethylamino)naphthalen-1-yl]sulfonylamino]-4-carboxybutanoyl]pyrrolidine-2-carbonyl]amino]-4-methylpentanoyl]amino]-3-phenylpropanoyl]amino]propanoyl]amino]-4-carboxybutanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-6-[[4-[[4-(dimethylamino)phenyl]diazenyl]benzoyl]amino]hexanoic acid

H-Glu(EDANS)-Pro-Leu-Phe-Ala-Glu-Arg-Lys(DABCYL)-OH; 1914987-47-5;

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)

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.)