A novel methylated analogue of L-Mimosine exerts its therapeutic potency through ROS production and ceramide-induced apoptosis in malignant melanoma.

Melanoma is an aggressive and highly metastatic type of skin cancer where the design of new therapies is of utmost importance for the clinical management of the disease. Thus, we have aimed to investigate the mode of action by which a novel methylated analogue of L-Mimosine (e.g., L-SK-4) exerts its therapeutic potency in an in vitro model of malignant melanoma. Cytotoxicity was assessed by the Alamar Blue assay, oxidative stress by commercially available kits, ROS generation, caspase 3/7 activation and mitochondrial membrane depolarisation by flow cytometry, expression of apoptosis-related proteins by western immunoblotting and profiling of lipid biosynthesis by a metabolomic approach. Overall, higher levels of ROS, sphingolipids and apoptosis were induced by L-SK-4 suggesting that the compound's therapeutic potency is mediated through elevated ROS levels which promote the upregulation of sphingolipid (ceramide) biosynthesis thus leading to the activation of both extrinsic and intrinsic apoptosis, in an experimental model of malignant melanoma.

Mimosine Dipeptide Enantiomsers: Improved Inhibitors against Melanogenesis and Cyclooxygenase.

Melanogenesis plays an important role in the protection of skin against UV through production of melanin pigments, but abnormal accumulation of this pigment causes unaesthetic hyperpigmentation. Much effort is being made to develop effective depigmenting agents. Here, we show for the first time that a small library of mimosine dipeptide enantiomers (Mi-L/D-amino acid) inhibit the melanogenesis in B16F10 melanoma cells by down-regulating the cellular tyrosinase with little effect on their growth or viability. Two of them, Mi-D-Trp and Mi-D-Val, turned out to be the most potent inhibitors on melanin content and cellular tyrosinase in B16F10 melanoma cells. In addition, most of the mimosine dipeptides were more potent than mimosine for inhibiting cyclooxygenase 1 (COX-1) with IC50 of 18-26 µM. Among them, Mi-L-Val and Mi-L-Trp inhibited cyclooxygenase 2 (COX-2) more potently than indomethacin, with IC50 values of 22 and 19 µM, respectively. Taken together, our results suggest the possibility that mimosine dipeptides could be better candidates (than mimosine) for anti-melanogenic (skin hyperpigmentation treatment) and cyclooxygenase (COX) inhibition.

Productive performance and urinary excretion of mimosine metabolites by hair sheep grazing in a silvopastoral system with high densities of Leucaena leucocephala.

The aim of this study was to evaluate daily weight gain (DWG), total dry matter (DM) intake, rumen degradability of forage, and urinary excretion of mimosine metabolites by hair sheep in a silvopastoral system with high densities of Leucaena leucocephala. A completely randomized design was carried out with two treatments: treatment 1 (T1) silvopastoral system with leucaena at a density of 35,000 plants/ha and treatment 2 (T2), leucaena at a density of 55,000 plants/ha. Leucaena was associated with tropical grasses Panicum maximum and Cynodon nlemfluensis. Twenty-four male Pelibuey lambs of 23.2 ± 3.4 kg live weight (LW) were used (12 lambs per treatment). Results showed differences (P < 0.05) in DWG of T1 (106.41 ± 11.66 g(-1) sheep(-1)) with respect to that of T2 (81.33 ± 11.81 g(-1) sheep). Voluntary intake was higher in lambs from T1 (83.81 ± 04.07 g DM/kg LW(0.75)) with respect to that from T2 (71.67 ± 8.12 g DM/kg LW(0.75)). There was a difference in color of urine between sheep of T1 and T2, the latter giving positive results for the presence of metabolites derived from mimosine (3-4 dihydroxypyridine and 2-3 dihydroxy pyridone). Rumen degradability of DM of L. leucocephala was higher (P < 0.05) compared to that of P. maximum and C. nlemfluensis (72.94 ± 0.40 vs. 67.06 ± 1.50 and 63.25 ± 1.51 %, respectively). It is concluded that grazing at high densities of L. leucocephala affects daily weight gain of hair sheep, possibly due to ingestion of high amounts of mimosine which may exert an adverse effect on voluntary intake.

Preparative scale isolation, purification and derivatization of mimosine, a non-proteinogenic amino acid.

Focusing on drug discovery non-proteinogenic amino acids have often been used as important building blocks for construction of compound libraries in the filed of combinatorial chemistry and chemical biology. Highly homogeneous L: -mimosine, α-amino-ß-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid, a non-proteinogenic amino acid, has been successfully isolated and purified on an industrial scale from wild leaves of Leucaena (Leucaena leucocephala de Wit) which is a widely distributed legume in Okinawa, a sub-tropical island in Japan. Optical purity determinations used for quality control have been established through diastereomer formation. Physico-chemical properties and biological properties of purified mimosine have been clarified. Mimosine is sparingly soluble in water and organic solvents but can be dissolved in aqueous alkaline solution. The tyrosinase pathway is of particular interest in the cosmetic field, since mimosine is an analog of tyrosine. Thus the present purified mimosine have been tested in tyrosinase inhibitory assays. The IC50 for tyrosinase inhibitory activity of purified Mim was compared with kojic acid. Mimosine shows significant inhibition of melanin production in murine melanoma cells. The derivatization of mimosine has been investigated with a focus on its use in conventional peptide syntheses to generate mimosyl peptides. N-(9-Fluorenylmethoxycarbonyloxy)-mimosine and resin-bound mimosine for solid-phase syntheses have also been performed. Highly homogeneous Mim is a useful material for the development of functional cosmetics or active pharmaceutical ingredients.

Selective determination of mimosine and its dihydroxypyridinyl derivative in plant systems.

Our observations on the growth stimulatory nature of mimosine, (beta-(3-hydroxy-4-pyridon-1-yl)-L-alanine), the toxic non-protein plant amino acid, in some model experimental systems, warranted sensitive and selective routine estimations. For the determination of both mimosine and DHP, an indirect spectrophotometric method was developed based on their individual reaction with known excess of DZSAM and by estimating the remaining DZSAM with N-(1-naphthyl)ethylene-diamine (NEDA). The resultant decrease in the secondary coupled product was measured at 540 nm. On equimolar basis, DHP had 40% of the reactivity of mimosine while interference from other relevant compounds was 15-35%. The determination of mimosine and DHP in tissue samples under different physiological conditions was effected after paper chromatographic separation of mimosine and DHP with distinctly differing Rf, from other compounds. The indirect method is superior in terms of absolute selectivity, sensitivity and ease of applicability with linear decreases in absorbance, proportional to increasing concentrations of mimosine from 0.1 to 0.75 microM or DHP from 0.2 to 1.5 microM and with recoveries of 99.2 to 100.5%.

Preparation and determination of structure of L-3-deoxymimosine-containing peptides.

L-3-Deoxymimosine-containing decapeptides were prepared for the development of protein tyrosine kinase (PTK) inhibitors. During the preparation of peptides, several side products were formed. Identification and determination of major peptides generated were reported.

Molecular recognition of synthetic siderophore analogues: a study with receptor-deficient and fhu(A-B) deletion mutants of Escherichia coli.

The biological activity of six synthetic siderophore analogues (two dihydroxamates, two trihydroxamates, one tetrahydroxamate and one 3-hydroxy-4(1H)pyridinone) has been studied in Escherichia coli, Morganella morganii 13 and Proteus mirabilis 8993 strains by using growth promotion tests. Various transport-deficient mutants of E. coli were used to study the route of entry into gram-negative bacteria. The results indicated that the synthetic hydroxamate compounds are transported via Fhu-mediated transport systems, although receptor specificity was low. This could be proven by using a delta (fhuA-B) E. coli mutant as a control in which growth promotion by natural hydroxamates was completely abolished, suggesting that a periplasmic binding-protein-dependent transport system (FhuB, C, D) is required for the transport of all synthetic ferric hydroxamate complexes. Although utilization of the synthetic hydroxamates was generally lower than that of the natural siderophores, differences in growth promotion could be detected. Highest activity was observed with the dihydroxamate DOCYDHAMA ligand which supported growth at concentrations < 1 mM. In comparison with other polyamino-polyhydroxamate ligands studied, this dihydroxamate ligand has an extra diamide backbone that could be important for the interaction with the receptors or with FhuD. The synthetic trihydroxamate and tetrahydroxamate ligands showed a relatively low siderophore activity. Studies with Proteus and Morganella in the presence of increasing bipyridyl concentrations showed a decreased growth promotion with the synthetic ferric hydroxamates, suggesting the involvement of a reduction step during iron mobilization or an increased toxicity of bipyridyl. This was not observed in the case of the 3-hydroxy-4(1H)pyridinone where bipyridyl had no effect.

Degradabilidade da proteína e desdobramento da mimosina da Leucaena leucocephala (lam) de with no rúmen de búfalos sob dietas com diferentes níveis de proteínas / Protein degradability and transformation of miimosine from Leucaena leucocephala (lam) the with in the rumen of buffaloes under diets with different protein levels

A técnica de sacos de náilon suspensos no rúmen foi utilizada para estudar a degradabilidade da proteína bruta e do desdobramento da mimosina da Leucaena leucocephala em búfalos submetidos a um experimento em quadrado latino (4 x 4) com diferentes níveis de proteína bruta na raçäo (8,7; 10,5; 12,5 e 14,9 por cento na MS). As raçöes näo promoveram diferenças significativas (P>0,01) na degradabilidade ruminal da proteína bruta da leucaena, que variou entre 30,92 por cento a 71,70 por cento em 3 e 48 horas de suspensäo no rúmen, respectivamente. Houve uma lenta degradaçäo ruminal da proteína e um acentuado desdobramento da mimosina nas amostras de leucaena colocadas dentro dos sacos de náilon no rúmen de búfalos

Depilatory effects of certain chemicals during the first hair growth cycle in sucking mice.

The chemicals were administered, subcutaneously, orally or topically. Generally, the depilation produced in the mice by mimosine or cyclophosphamide differed from that produced by the steroid analogues tested. In the first 2 cases almost completely naked mice were commonly seen, while in the steroid-treated groups the complete inhibition of all hair fibres was rare. This is discussed in relation to the effects of the same compounds on wool growth in sheep. When related to bodyweight, the doses of cyclophosphamide (62 mg/kg0.75) and dexamethasone (5--10 mg/kg0.75), that depilated mice in our experiments were in good agreement with those reported to inhibit the growth of wool fibres in some sheep. An example of synergism in depilatory effect between dexamethasone and cyclophosphamide is also presented. The time of onset and the initial spread over the body of the 2nd hair cycle in depilated mice was observed.

Inhibition of wool follicle DNA synthesis by mimosine and related 4(1H)-pyridones.

The in vitro incorporation of [3H]thymidine has been examined in thin slices of sheep skin. Most of the radioactivity (88%) was incorporated into the bulb cells of the wool follicles, and the technique is therefore suitable for the study of some aspects of wool follicle DNA synthesis. The effect of mimosine and a number of related 4(1H)-pyridones on [3H]thymidine incorporation into sheep skin slices was examined. Mimosine was shown to inhibit the incorporation at a concentration of 0-2 mM. At this concentration, the incorporation of [3H]uridine or [14C]leucine was not affected. The inhibition of [3H]thymidine incorporation was time dependent, 2 h of incubation being required for maximal inhibition of DNA synthesis, and was readily reversible by removal of mimosine from the incubation medium. The 3-hydroxyl-4-oxo function of the pyridone ring appears to be directly involved in DNA synthesis inhibition. The amino acid side chain is not a toxophoric centre, but changes in its polarity have been shown to affect the inhibitory activity. The results suggest that the primary action of mimosine on the inhibition of wool biosynthesis in vivo is the inhibition of follicle bulb cell DNA synthesis and consequently of cell division.