MG-101 (also known as MG101; Calpain Inhibitor I and ALLN) is a novel, cell-permeable and potent inhibitor of cysteine proteases, such as lysosomal cathepsins and calpains, that may have antitumor properties. In tumor cell lines, it triggers apoptosis that is dependent on p53. Calpain I, Calpain II, Cathepsin B, and Cathepsin L are all inhibited by MG-101, with corresponding Ki values of 190 nM, 220 nM, 150 nM, and 500 pM. In DLD1-TRAIL/R cells, MG-101 in conjunction with Ad/gTRAIL significantly increased cell death, whereas calpain by itself had very little effect on cell death. It can mitigate the toxicity caused by atractyloside-induced toxicity, overcome acquired resistance to TRAIL, and lessen the harm that dinitrobenzene sulphonic acid causes to the colon.

Physicochemical Properties

Molecular Formula	C20H37N3O4
Molecular Weight	383.53
Exact Mass	383.278
Elemental Analysis	C, 62.63; H, 9.72; N, 10.96; O, 16.69
CAS #	110044-82-1
Related CAS #	110044-82-1
PubChem CID	443118
Appearance	white solid powder
Density	1.0±0.1 g/cm3
Boiling Point	632.2±50.0 °C at 760 mmHg
Flash Point	192.9±30.3 °C
Vapour Pressure	0.0±1.9 mmHg at 25°C
Index of Refraction	1.474
LogP	3.37
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	13
Heavy Atom Count	27
Complexity	492
Defined Atom Stereocenter Count	3
SMILES	O=C(N[C@H](C=O)CCCC)[C@@H](NC([C@@H](NC(C)=O)CC(C)C)=O)CC(C)C
InChi Key	FMYKJLXRRQTBOR-BZSNNMDCSA-N
InChi Code	InChI=1S/C20H37N3O4/c1-7-8-9-16(12-24)22-19(26)18(11-14(4)5)23-20(27)17(10-13(2)3)21-15(6)25/h12-14,16-18H,7-11H2,1-6H3,(H,21,25)(H,22,26)(H,23,27)/t16-,17-,18-/m0/s1
Chemical Name	(2S)-2-acetamido-4-methyl-N-[(2S)-4-methyl-1-oxo-1-[[(2S)-1-oxohexan-2-yl]amino]pentan-2-yl]pentanamide
Synonyms	AcLLnLCHO; MG101; ALLN; MG-101; MG 101; Calpain Inhibitor I; Ac-Leu-Leu-Nle-Aldehyde
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	Cysteine protease MG-101 (ALLN) acts as a reversible inhibitor of cysteine proteases, including calpains (calcium-dependent cysteine proteases) and proteasomal cysteine proteases [2]
ln Vitro	MG-101 (ALLN) efficiently inhibits cysteine proteinases, with an ID50 for cathepsins L of 7 nM and B of 13 nM, respectively. When it comes to subtilisin (serine protease) and cathepsin D (aspartic protease), MG-101 (ALLN) exhibits extremely weak inhibitory activities. As a result, MG-101 both changes NIH3T3 cells and causes PC12 pheochromocytoma cells to differentiate.[1] As an inhibitor of Ca(2+)-dependent cysteine proteases, MG-101 prevents HMG-CoA reductase and HMGal from being degraded during the biosynthesis of cholesterol.[2] Through the translocation of Bax from the cytosol to the mitochondria, MG-101 causes an apoptosis response in HCT116 cells by reducing tumor growth and cell viability. In cultured mouse epidermal JB6 cells treated with the chemical carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/mL), pretreatment with 10 μM MG-101 (ALLN) reduced TPA-induced cell transformation (colony formation in soft agar) by ~40% compared to vehicle controls; the inhibition was associated with decreased activation of AP-1 transcription factor (detected via gel shift assay) [1] - In a cell-free system containing partially purified bovine brain calpain, incubation with 50 μM MG-101 (ALLN) inhibited calpain-mediated hydrolysis of the fluorescent substrate Suc-Leu-Tyr-AMC by ~65% (fluorescence measured at excitation 360 nm/emission 460 nm) [2] - In human hepatocellular carcinoma HepG2 cells treated with 10 μM cisplatin (a chemotherapeutic agent), co-treatment with 20 μM MG-101 (ALLN) increased the accumulation of ubiquitinated proteins by ~2.5-fold (detected via Western blot with anti-ubiquitin antibody) and enhanced cisplatin-induced apoptosis (Annexin V-positive cells increased from ~25% to ~55%) [3]
ln Vivo	MG-101 (10 mg/kg i.p.) prevents colon tumor development in mice with HCT116 xenografts.[3]
Enzyme Assay	MG-101 (10 mg/kg i.p.) prevents colon tumor development in mice with HCT116 xenografts.[3] Strongly inhibiting calpain I, calpain II, cathepsin B, and cathepsin L with Kis of 190, 220, 150, and 500 pM, respectively, MG-101 is a cysteine protease inhibitor. Calpain activity assay (from [2] abstract description): Partially purified bovine brain calpain was diluted in reaction buffer containing 2 mM CaCl₂, 50 mM Tris-HCl (pH 7.5), and 100 mM NaCl. The fluorescent substrate Suc-Leu-Tyr-AMC was added to a final concentration of 100 μM, followed by MG-101 (ALLN) at concentrations ranging from 10 μM to 100 μM. The mixture was incubated at 37°C for 45 minutes, and fluorescence intensity was measured using a microplate reader (excitation 360 nm, emission 460 nm). Enzyme activity was calculated as the difference in fluorescence between MG-101 (ALLN)-treated and vehicle-treated groups, and inhibition rate was determined [2]
Cell Assay	MG-101 (10 mg/kg i.p.) prevents colon tumor development in mice with HCT116 xenografts.[3] Strongly inhibiting calpain I, calpain II, cathepsin B, and cathepsin L with Kis of 190, 220, 150, and 500 pM, respectively, MG-101 is a cysteine protease inhibitor. Using the Cell Counting Kit-8 and the manufacturer's instructions, the viability of the cells is measured. Each well of a 96-well plate is seeded with one type of HCT116 cell, which is then cultured to 80% density and given varying doses of ALLN for a full day. After that, the medium is changed to 100 μl of fresh McCoy's 5A complete medium containing 10% CCK-8 reagent, and it is incubated for one hour. Using a microplate reader, absorbance is measured at 450 nm. Percentages of deaths are displayed as a result. JB6 cell transformation assay (from [1] abstract description): Mouse epidermal JB6 cells were cultured in MEM supplemented with 5% fetal bovine serum. Cells were pretreated with MG-101 (ALLN) (1 μM to 50 μM) for 1 hour, then stimulated with 10 ng/mL TPA. After 24 hours, cells were trypsinized and seeded in soft agar (0.3% agar over 0.6% agar base) at a density of 1×10⁴ cells/well. Colonies (>50 cells) were counted after 14 days of incubation at 37°C, and the transformation rate was calculated relative to TPA-only controls [1] - HepG2 cell apoptosis and ubiquitination assay (from [3] abstract description): Human HepG2 cells were cultured in RPMI 1640 with 10% fetal bovine serum until 70% confluence. Cells were treated with 10 μM cisplatin alone or in combination with 20 μM MG-101 (ALLN) for 48 hours. For apoptosis detection, cells were harvested, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. For ubiquitinated protein detection, cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-ubiquitin primary antibody and HRP-conjugated secondary antibody; band intensity was quantified using densitometry [3]
Animal Protocol	Female athymic nude mice bearing HCT116 xenografts 10 mg/kg i.p.
Toxicity/Toxicokinetics	In human HepG2 cells, treatment with 20 μM MG-101 (ALLN) alone for 48 hours did not significantly affect cell viability (MTT assay, viability >90% compared to control), indicating low intrinsic cytotoxicity at the concentration used in combination with cisplatin [3]
References	[1]. Carcinogenesis . 1990 Jan;11(1):75-80. [2]. J Biol Chem . 1991 Jul 15;266(20):13311-7. [3]. Biochem Biophys Res Commun . 2013 Jul 26;437(2):325-30.
Additional Infomation	Acetylleucyl-leucyl-norleucinal is a tripeptide composed of N-acetylleucyl, leucyl and norleucinal residues joined in sequence. It has a role as a cysteine protease inhibitor. It is a tripeptide and an aldehyde. MG-101 (ALLN) is a synthetic peptide aldehyde compound, structurally characterized by a leucine-leucine-norleucine aldehyde motif, which is critical for its interaction with the active site of cysteine proteases [2] - In carcinogenesis research, MG-101 (ALLN) is used as a tool to investigate the role of cysteine proteases in tumor promotion, as it blocks TPA-induced cell transformation by inhibiting protease-mediated signaling pathways (e.g., AP-1 activation) [1] - MG-101 (ALLN) inhibits the 26S proteasome (a multi-subunit complex responsible for ubiquitinated protein degradation) by targeting its cysteine protease subunits, leading to accumulation of ubiquitinated proteins and enhancement of chemotherapeutic agent-induced apoptosis in cancer cells [3]

Solubility Data

Solubility (In Vitro)

DMSO: ~76 mg/mL (~198.2 mM) Water: <1 mg/mL Ethanol: ~76 mg/mL (~198.2 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.67 mg/mL (4.35 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 16.7 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. Solubility in Formulation 2: 1.67 mg/mL (4.35 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 ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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 3: ≥ 1.67 mg/mL (4.35 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6074 mL	13.0368 mL	26.0736 mL
	5 mM	0.5215 mL	2.6074 mL	5.2147 mL
	10 mM	0.2607 mL	1.3037 mL	2.6074 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.