LRRK2-mediated Rab10 and Rab12 phosphorylation
Dipeptidyl peptidase I (DPPI, also known as cathepsin C). [2]

In MEF and A549 cells, LRRK2-mediated phosphorylation of Rab10 and Rab12 is enhanced by L-leucyl-L-leucine methyl ester hydrobromide (1 mM; 0.5-2 h) [3]. CD4-lymphocytes hydrolyze L-leucyl-L-leucine methyl ester hydrobromide (10-250 μM; 15 minutes) to an insoluble CCI3COOH product [2].

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L-leucyl-L-leucine methyl ester (250 µM) exposure of human peripheral blood lymphocytes for 15 min at 22°C followed by 18 hr culture at 37°C resulted in loss of all CD16+ NK cells and majority of CD8+ T cells, with reciprocal enrichment of CD4+ T cells; all NK cell function (against K562 targets) and capacity to generate allospecific cytotoxic T lymphocytes were ablated [2].
Preincubation with 10 µM Gly-Phe-CHN2 (specific DPPI inhibitor) for 1 hr at 37°C prevented L-leucyl-L-leucine methyl ester-induced depletion of CD16+ and CD8+ cells and preserved NK and CTL function [2].
Incubation of T8/NK-enriched lymphocytes with 100-250 µM L-leucyl-L-leucine methyl ester led to production of CCl3COOH-insoluble metabolites (polymers of structure (Leu-Leu)n-OMe, n≥3 such as Leu4-OMe, Leu6-OMe, Leu6), whereas 10 µM produced none [2].
Leu6-OMe (but not Leu2-OMe, Leu4-OMe, or non-esterified peptides) caused significant lysis of human red blood cells; addition of exogenous DPPI plus ≥250 µM L-leucyl-L-leucine methyl ester also induced RBC lysis, while DPPI or drug alone had no effect [2].
Cycloheximide did not inhibit generation of CCl3COOH-insoluble product; iodoacetamide and Gly-Phe-CHN2 blocked it; NH4Cl increased the amount of insoluble product [2].

In murine models of bone marrow transplantation across multiple MHC and non-MHC barriers, donor cells incubated with 250 µM L-leucyl-L-leucine methyl ester for 15 min at 22°C, then washed, prevented in vivo development of donor anti-host CTL and lethal graft-versus-host disease while permitting rapid engraftment and establishment of long-lived immunocompetent chimeras [2].

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].

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DPPI activity was assayed fluorometrically at pH 5.0 in 0.05 M sodium acetate buffer containing 10 mM cysteine, 30 mM NaCl, and 1 mM EDTA, using Gly-Phe-β-naphthylamide as substrate [2].
Cathepsin B was assayed at pH 6.3 in phosphate buffer with 1 mM EDTA and 10 mM cysteine, using (Z)-Arg-Arg-β-naphthylamide as substrate [2].
Inhibition of DPPI was assessed using Gly-Phe-CHN2; preincubation of cells with 1-100 µM Gly-Phe-CHN2 for 60 min led to >95% inhibition of intracellular DPPI activity without significantly altering cathepsin B activity [2].

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].

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No detailed cell assay protocol (e.g., cell viability, Western blot, PCR) is provided. The following summary is from the text: For NK cell cytotoxicity, a 4 hr 51Cr-release assay against K562 target cells was used; percent specific cytotoxicity and lytic units were calculated [2].
For allospecific CTL activity, lymphocytes were cultured for 6 days with irradiated allogeneic stimulator cells and then assayed in a 4 hr 51Cr-release assay [2].
To measure uptake and metabolism, T8/NK-enriched lymphocytes (2.5×10^6 cells/mL) in PBS were incubated with [3H]L-leucyl-L-leucine methyl ester and unlabeled drug at 22°C for 15 min, then centrifuged through silicone oil or washed and cultured at 37°C in RPMI 1640 with 0.5% bovine serum albumin. An equal volume of 20% CCl3COOH was added, centrifuged, precipitates washed, dried under vacuum, resuspended in anhydrous methanol, and analyzed by two-dimensional TLC on silica gel with chloroform/methanol/acetic acid/butanol (36:1:8:4) [2].

Donor bone marrow or spleen cells were incubated with 250 µM L-leucyl-L-leucine methyl ester for 15 min at 22°C, washed, and then transplanted into irradiated recipient mice across multiple MHC and non-MHC histocompatibility barriers. No other in vivo dosing or formulation details are provided [2].

L-leucyl-L-leucine methyl ester is toxic to cytotoxic lymphocytes (NK cells, CD8+ T cells, CD4+ allospecific CTL) and myeloid cells (monocytes, polymorphonuclear leukocytes, myeloid tumor cell lines), while helper T cells, B cells, and non-bone-marrow-derived cell lines (e.g., endothelial cells, fibroblasts, renal carcinoma cells) are completely resistant [2].
Toxicity is concentration-dependent: exposure to 10-20 µM causes no adverse effects, whereas 100-250 µM ablates all NK and CTL function [2].
Human erythrocytes (which lack detectable DPPI) are not lysed by L-leucyl-L-leucine methyl ester alone, but are lysed by the DPPI-catalyzed polymerization product Leu6-OMe or by the combination of exogenous DPPI plus L-leucyl-L-leucine methyl ester [2].

[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.

Exposure 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]

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L-leucyl-L-leucine methyl ester acts as a pro-drug that is converted by DPPI (cathepsin C) via its acyltransferase activity into (Leu-Leu)n-OMe polymers (n≥3) with detergent-like membranolytic properties. This conversion occurs within the lysosomal granules of cytotoxic lymphocytes, which contain 10- to 20-fold higher levels of DPPI than resistant cell types (e.g., CD16+ NK cells: 2.54 mM substrate cleaved/µg protein/hr; CD4+ T cells: 0.19; CD19+ B cells: 0.13; CD8+ T cells: 0.62) [2].
The selective toxicity is attributed to the high DPPI content in cytolytic cells; other characteristics such as acidification capacity or presence of other proteases may also contribute [2].
The drug has been used to delete cytotoxic lymphocyte responses in vitro and to prevent graft-versus-host disease in murine bone marrow transplantation models [2].

C13H26N2O3.HBR

339.26908

338.121

16689-14-8

6491-83-4 (HCl)

73553531

H-Leu-Leu-OMe.HBr ;L-leucyl-L-leucine methyl ester hydrobromide

LL

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

3.113

3

4

8

19

277

2

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

QIPBCIHWFATZOF-ACMTZBLWSA-N

InChI=1S/C13H26N2O3.BrH/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;hydrobromide

LLOMe hydrobromide; Leu-Leu methyl ester hydrobromide; 16689-14-8; Leu-Leu methyl ester hydrobromide; Leu-Leu-ome HBr; Leu-Leu-ome hydrobromide; 48TF27RT8L; L-Leucine, L-leucyl-, methyl ester, monohydrobromide; Methyl leucylleucinate hydrobromide; L-Leucyl-L-Leucine methyl ester (hydrobromide); H-Leu-Leu-OMe hydrobromide

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 : ~125 mg/mL (~368.44 mM)
H2O : ~100 mg/mL (~294.75 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.13 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 (6.13 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 (6.13 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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Solubility in Formulation 4: 100 mg/mL (294.75 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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