LRRK2-mediated Rab10 and Rab12 phosphorylation
Methyl leucylleucinate HCl targets lymphocytes with cytotoxic potential, selectively eliminating them. Its mechanism involves inducing lysosomal pathway stress. It enhances LRRK2-mediated Rab10 and Rab12 phosphorylation, which is involved in Parkinson's disease research. In MEFs and A549 cells, LRRK2-mediated Rab10 and Rab12 phosphorylation is enhanced by this compound.

In MEFs and A549 cells, LRRK2-mediated Rab10 and Rab12 phosphorylation is enhanced by L-leucyl-L-leucine methyl ester (1 mM; 0.5-2 h)[3]. CD4-lymphocytes convert L-leucyl-L-leucine methyl ester (10-250 μM; 15 min) into an insoluble CCI3COOH product[2].
In vitro, Methyl leucylleucinate HCl is used to study the lysosomal pathway and its effects on immune cells. It enhances LRRK2-mediated Rab10 and Rab12 phosphorylation in MEFs and A549 cells at concentrations of 1 mM. It is converted by CD4-lymphocytes into an insoluble product. Its effects on cell viability and immune function are measured in cell-based assays.

Methyl leucylleucinate HCl is not used for in vivo therapeutic applications. It is a research tool for studying lysosomal function and immune cell biology. Its selectivity for lymphocytes with cytotoxic potential has been studied in vitro. It can induce lysosomal pathway stress, which is relevant to Parkinson's disease research.

Mutations that enhance LRRK2 protein kinase activity cause inherited Parkinson's disease. LRRK2 phosphorylates a group of Rab GTPase proteins, including Rab10 and Rab12, within the effector-binding switch-II motif. Previous work has indicated that the PARK16 locus, which harbors the gene encoding for Rab29, is involved in Parkinson's, and that Rab29 operates in a common pathway with LRRK2. Co-expression of Rab29 and LRRK2 stimulates LRRK2 activity by recruiting LRRK2 to the surface of the trans Golgi network. Here, we report that knock-out of Rab29 does not influence endogenous LRRK2 activity, based on the assessment of Rab10 and Rab12 phosphorylation, in wild-type LRRK2, LRRK2[R1441C] or VPS35[D620N] knock-in mouse tissues and primary cell lines, including brain extracts and embryonic fibroblasts. We find that in brain extracts, Rab12 phosphorylation is more robustly impacted by LRRK2 inhibitors and pathogenic mutations than Rab10 phosphorylation. Transgenic overexpression of Rab29 in a mouse model was also insufficient to stimulate basal LRRK2 activity. We observed that stimulation of Rab10 and Rab12 phosphorylation induced by agents that stress the endolysosomal system (nigericin, monensin, chloroquine and LLOMe) is suppressed by LRRK2 inhibitors but not blocked in Rab29 deficient cells. From the agents tested, nigericin induced the greatest increase in Rab10 and Rab12 phosphorylation (5 to 9-fold). Our findings indicate that basal, pathogenic, as well as nigericin and monensin stimulated LRRK2 pathway activity is not controlled by Rab29. Further work is required to establish how LRRK2 activity is regulated, and whether other Rab proteins can control LRRK2 by targeting it to diverse membranes[3].
The in vitro activity of Methyl leucylleucinate HCl can be assessed using cell-based assays. For example, its ability to enhance LRRK2-mediated phosphorylation of Rab10 and Rab12 can be measured by Western blot in MEFs and A549 cells. Its conversion by CD4-lymphocytes can be measured by the formation of an insoluble product.

L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe), a dipeptide condensation product of L-leucine methyl ester generated by human monocytes (M phi) or polymorphonuclear leukocytes, eliminates all natural killer cell (NK) function from mixed lymphocyte populations. In the present studies, the specificity of the action of Leu-Leu-OMe was examined. It was found that a variety of tissue culture cells and tumor lines of nonlymphoid origin were completely resistant to any demonstrable Leu-Leu-OMe-mediated toxicity. Furthermore, the erythroleukemia line K562, the T cell line Molt-4, the B cell lines HS-Sultan and Daudi, and EBV-transformed B cell lines were unaffected by concentrations of this compound that completely eliminated NK cells. Similarly, the vast majority of OKT4+ lymphocytes manifested no significant toxicity after Leu-Leu-OMe exposure. Furthermore, they retained the capacity to proliferate normally in response to allogeneic cells as well as the ability to provide help for the generation of immunoglobulin-secreting cells (ISC). However, Leu-Leu-OMe caused partial depletion of OKT8+ cells from mixed populations of lymphocytes. After such exposure, the remaining OKT8+ cells were still capable of proliferating in mixed lymphocyte cultures, but the suppressive effect of these cells on ISC generation was abolished. Furthermore, both precursors and activated effectors of cytotoxic T lymphocyte (CTL) and activated NK-like activity generated in mixed lymphocyte cultures were eliminated by exposure to low concentrations of Leu-Leu-OMe. Indeed, both OKT4+ and OKT8+ CTL were eliminated by Leu-Leu-OMe. In addition, both peripheral blood M phi and U937 cells, a human cell line with many M phi-like characteristics, were sensitive to Leu-Leu-OMe-mediated toxicity, although only at two- to fivefold higher concentrations than those completely eliminating NK cells. These findings indicate that Leu-Leu-OMe has selective toxicity for NK cells, CTL, and M phi without adverse effects on a variety of other lymphoid or nonlymphoid cell types[1].
In vitro cellular assays for Methyl leucylleucinate HCl involve treating immune cells, such as lymphocytes or monocytes, with the compound and measuring cell viability, apoptosis, or the induction of lysosomal stress. Its effects on LRRK2-mediated phosphorylation are measured in MEFs and A549 cells.

Methyl leucylleucinate HCl is not typically used in in vivo animal experiments as a standalone compound. However, it may be used in research models of lysosomal storage diseases or Parkinson's disease to study the effects of lysosomal stress. Its effects on LRRK2-mediated phosphorylation have been studied in mouse tissues.

Methyl leucylleucinate HCl has limited pharmacokinetic data available, as it is a research tool rather than a therapeutic agent. It is typically stored as a powder at room temperature. Its stability and solubility in water and DMSO are known.

Methyl leucylleucinate HCl is a research chemical and should be handled with standard laboratory precautions. It may be irritating to the skin, eyes, and respiratory tract. It is for research use only and is not intended for human use. Its toxicity is related to its ability to induce lysosomal stress in cells.

[1]. The immunosuppressive activity of L-leucyl-L-leucine methyl ester: selective ablation of cytotoxic lymphocytes and monocytes. J Immunol. 1986 Feb 1;136(3):1038-48.
[2]. Mechanism of L-leucyl-L-leucine methyl ester-mediated killing of cytotoxic lymphocytes: dependence on a lysosomal thiol protease, dipeptidyl peptidase I, that is enriched in these cells. Proc Natl Acad Sci U S A. 1990 Jan;87(1):83-7.
[3]. Endogenous Rab29 does not impact basal or stimulated LRRK2 pathway activity. Biochem J. 2020 Nov 27;477(22):4397-4423.

Exposing mouse or human lymphocytes to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) leads to selective death of cytotoxic lymphocytes while helper T cells and B cells remain functional. Cytotoxic lymphocytes incubated in toxic concentrations of Leu-Leu-OMe have been found to contain membrane-lysing metabolites of the structure (Leu-Leu)n-OMe, where n ≥ 3. The sensitivity of cytotoxic lymphocytes to Leu-Leu-OMe depends on these metabolites produced by lysosomal thiol proteases—dipeptidyl peptidase I. Dipeptidyl peptidase I is present in cytotoxic lymphocytes at much higher levels than in cells without cytolytic potential or of non-myeloid origin. Therefore, this granzyme is essential for the unique role of Leu-Leu-OMe and may provide a target for the development of other immunotherapies aimed at eliminating cytotoxic lymphocyte responses. [2]
Methyl leucylleucinate HCl is also known by synonyms including H-LEU-LEU-OME HCl, Leu-Leu-ome HCl, and L-Leucyl-L-Leucine methyl ester hydrochloride. It is a research tool used to study lysosomal pathways, immune cell function, and Parkinson's disease. It is not an FDA-approved drug and is exclusively for research purposes.

C13H27CLN2O3

294.818083047867

294.171

C, 52.96; H, 9.23; Cl, 12.02; N, 9.50; O, 16.28

6491-83-4

16689-14-8 (HBr)

15598024

H-Leu-Leu-OMe.HCl; L-leucyl-L-leucine methyl ester hydrochloride

LL

Typically exists as white to off-white solids at room temperature

171-181 °C

2.956

3

4

8

19

277

2

Cl.O=C([C@H](CC(C)C)N)N[C@H](C(=O)OC)CC(C)C

RVXQFTNCULOWRV-ACMTZBLWSA-N

InChI=1S/C13H26N2O3.ClH/c1-8(2)6-10(14)12(16)15-11(7-9(3)4)13(17)18-5;/h8-11H,6-7,14H2,1-5H3,(H,15,16);1H/t10-,11-;/m0./s1

methyl (2S)-2-[[(2S)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoate;hydrochloride

6491-83-4; H-LEU-LEU-OME HCL; Leu-Leu-ome HCl; H-Leu-leu-OMe hydrochloride; Leu-Leu-ome hydrochloride; Methyl leucylleucinate HCl; L-Leucyl-L-Leucine methyl ester (hydrochloride); 76R0TP2LNW; 76R0TP2LNW; Methyl leucylleucinate hydrochloride; UNII-76R0TP2LNW; L-Leucine, L-leucyl-, methyl ester, hydrochloride (1:1); ...; 6491-83-4;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~847.98 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.06 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.06 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.06 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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