bestatin (as bestatin methyl ester, BME) targets Zn²⁺-binding aminopeptidases, specifically puromycin-sensitive aminopeptidase A (PsaA) in Dictyostelium discoideum. BME is a more cell-permeable analog of bestatin.
bestatin also targets the related aminopeptidase PsaB (not characterized in this study). [4]

In ATRA-sensitive APL NB4 cells, bestatin promotes ATRA-induced differentiation and prevents ATRA-driven p38 MAPK phosphorylation. ATRA-resistant APL MR2 cells' differentiation block was not reversed by bestatin. When CD13 binds to the anti-CD13 antibody WM-15, p38 MAPK is phosphorylated. This decreases Bestatin's suppression of p38 MAPK phosphorylation and totally removes Bestatin's stimulatory effect on ATRA-induced NB4 cell differentiation [2]. Cells treated with bestatin (600 μM) underwent slower cell cycle progression because their frequency of cell division and growth were decreased. Bestatin suppresses D's intrinsic multinucleation and mitotic frequency. discoideum and does not cause D cell death. discoideum cells at 0-600 μM doses. In PsaA-GFP and GFP-expressing cell lysates, bestatin reduced the aminopeptidase activity by 69.39% and 39.93% of the control, respectively [4].

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In Dictyostelium discoideum, treatment with bestatin methyl ester (BME) at concentrations of 10, 50, 100, 300, or 600 μM for 48 h resulted in a concentration-dependent decrease in cell density increase. At 600 μM, the fold increase in cell density was 3.25-fold compared to 18.46-fold in untreated controls, representing 82.3% inhibition.
BME treatment increased cell doubling time in a concentration-dependent manner, from 8.22 ± 0.96 h at 0 μM to 30.41 ± 3.07 h at 600 μM (3.70-fold increase).
BME-treated cells (600 μM) had nucleoli 0.23-fold smaller (4.69% ± 0.93% of nuclear area) than untreated cells (20.07% ± 2.93%), a phenomenon associated with slow growth.
BME treatment increased the proportion of mononucleated cells. At 600 μM, mononucleated cells were 86.67% ± 6.54% compared to 38.89% ± 4.81% in untreated controls (2.23-fold increase).
BME inhibited nuclear concentration increase. After 48 h, nuclear concentration in 600 μM BME-treated culture was 0.11-fold (89% inhibition) of untreated culture.
BME was not cytotoxic; at 300 μM and 600 μM, viable cells were 99.14% ± 1.22% and 95.78% ± 3.25%, respectively, and cells in all mitotic stages were observed.
In developing cells, BME (600 μM and 900 μM) affected spore cell differentiation. At 600 μM, spores were rounder; at 900 μM, terminal spore cell differentiation was completely inhibited (no terminally differentiated spores in spore mass).
In PsaA-GFP-overexpressing cells, BME (600 and 900 μM) caused abnormal development: no fruiting bodies formed, cells aggregated into irregular mounds, and all cells terminally differentiated into stalk cells (no spores). In PsaAΔNLS2-GFP-overexpressing cells, BME (600 μM) caused rounded spores, and 900 μM inhibited terminal spore differentiation. [4]

In diabetic mice, bestatin (20 μM) dramatically suppressed MMP-9-specific sol zone density and considerably decreased CD13 expression as compared to mice treated with diabetic vehicles. Diabetes-stricken mice's VEGF and heparanase expression were markedly suppressed by bestatin therapy. In the retina of diabetic mice, intravitreal betastatin therapy markedly reduced the production of VEGF and HIF-1α. Furthermore, the elevation of heparanase expression in diabetic mice's retina can be considerably suppressed by intravitreal betastatin injection [1]. When splenocytes are treated with Bestatin (10, 1, and 0.1 mg/kg, i.p.), they produce more hemolytic anti-SRBC antibodies (PFC) and more 2-ME-resistant serum before antigen-enhancing humoral responses to SRBC occur. Hemagglutinin titer (0.1 mg/kg) in dosage. Injecting mice with Bestin (1 and 0.1 mg/kg) five times every other day following cyclophosphamide injection did not alter the drug's inhibitory effect on the number of PFC and, in fact, produced a greater decrease in total anti-SRBC hemagglutinin at the antigen dosage. 1 mg/kg seven days following stimulation [3].
In vivo development assay: Dictyostelium discoideum AX3 cells were allowed to develop on black membrane filters with filter pads soaked in distilled water containing 600 or 900 μM bestatin methyl ester (BME). Development proceeded for 24 h. Fruiting bodies were stained with calcofluor to analyze stalk and spore cell morphology. BME treatment inhibited spore cell differentiation and caused rounding of spores. At 900 μM, terminal spore differentiation was blocked. [4]

Fluorometric aminopeptidase assay: Cells were harvested, washed, and lysed in NP-40 lysis buffer. Total cell protein was quantified using Bradford assay. Ten microliters of total cell protein (1 mg/ml) was mixed with 290 μl of substrate solution containing 0.1 mg/ml dithiothreitol (DTT), 0.1 mg/ml albumin, and 1 mM alanine-β-naphthylamide. Fluorometric measurements were taken at 340 nm excitation and 400 nm emission after 15 and 30 minutes. The slope between the 15- and 30-minute measurements represented aminopeptidase activity. Total cell protein was preincubated with bestatin, amastatin, puromycin, EDTA, and/or ZnCl₂ for 20 minutes before the assay.
In this assay, whole-cell lysates of PsaA-GFP-overexpressing cells had over 2-fold (229.97% ± 41.3%) more aminopeptidase activity than parental AX3 cells. Lysates of GFP-only cells had similar activity (95.33% ± 6.2%) to AX3. Puromycin (200 μM) inhibited aminopeptidase activity in PsaA-GFP lysates by 39.39% ± 3.1% of control; in GFP lysates by 17.68% ± 1.9%. Bestatin methyl ester (BME, 300 μM) inhibited activity in PsaA-GFP lysates by 69.39% ± 10.5% of control, and in GFP lysates by 39.93% ± 18.7%. Amastatin (20 μM) inhibited PsaA-GFP lysates by 37.24% ± 3.6% and GFP lysates by 47.01% ± 12.2%. EDTA (5 mM) inhibited PsaA-GFP lysates by 43.00% ± 4.7% and GFP lysates by 14.90% ± 19.0%. ZnCl₂ (10 μM) partially rescued EDTA inhibition in PsaA-GFP lysates (17.68% rescue) and enhanced activity in GFP lysates by 27.24% ± 16.0%. ZnCl₂ alone enhanced activity in PsaA-GFP lysates by 85.18% ± 19.4% and in GFP lysates by 49.35% ± 18.0%. [4]

Cell viability staining: Cells were harvested after treatment with different concentrations of bestatin methyl ester (BME) for 24 hours, then stained with fluorescein diacetate (20 μg/ml) and propidium iodide for 5 minutes. At least 100 cells were counted for each treatment. Live cells (FDA-positive, PI-negative) were quantified. At 300 μM and 600 μM BME, viability was 99.14% ± 1.22% and 95.78% ± 3.25%, respectively, with no dying cells detected at lower concentrations.
Cell growth and doubling time assay: Growing Dictyostelium discoideum cells (1×10⁶ to 2×10⁶ cells/ml) were diluted to 1.0×10³ cells/ml and transferred to 12-well plates. Cells were treated with 0, 10, 50, 100, 300, or 600 μM BME and grown at 21°C with shaking at 180 rpm for 48 h. Cell density was measured using a hemocytometer at 0, 24, and 48 h. Doubling time was calculated from data between 24 and 48 h.
Nuclear counting: Cells were fixed in ultracold methanol and mounted with DAPI-containing antifade. Nuclei were counted using fluorescence microscopy.
Immunolocalization: Cells were fixed, blocked, and incubated with anti-PsaA or anti-NumA1 antibodies (1:20 or 1:40), followed by Alexa Fluor secondary antibodies. Cells were then incubated with anti-α-tubulin (1:100), followed by secondary antibody. Coverslips were mounted with DAPI-containing antifade. Images were captured using a Nikon Eclipse 50i microscope.
Western blot analysis: For analysis of PsaA expression, 2×10⁶ growing and developing cells were lysed with NP-40 lysis buffer. Samples (25 μg/lane) were separated on 12% SDS-PAGE, transferred to PVDF membrane, and probed with rabbit anti-PsaA (1:500), mouse anti-α-tubulin (1:1,000), or mouse anti-GFP (1:600). Blots were developed with ECL Plus detection system.
Northern blotting: Total RNA from growing and developing cells was separated on 1% agarose-formaldehyde gel (25 μg/lane), transferred to positively charged nylon membrane, and UV-crosslinked. DIG-labeled probes for psaA and rnlA were used. Prehybridization (2 h) and hybridization (overnight) at 42°C. Blots were exposed to film for 5 min.
Morphogen treatment: AX3 cells were starved in KK2 buffer at 2×10⁶ cells/ml, treated with 20 mM NH₄Cl, 1 mM cAMP, or 100 nM DIF-1 alone or in combination for up to 4 h with shaking at 250 rpm. Cells were harvested at 2 and 4 h post-treatment, lysed, and analyzed by Western blot for PsaA expression. [4]

Animal protocol not applicable as this study was performed in Dictyostelium discoideum, a social amoeba, not in animals. The "in vivo" experiments refer to development assays on filters using the organism. No animal (mammalian) protocols were described. [4]

[1]. Protective effects of bestatin in the retina of streptozotocin-induced diabetic mice. Exp Eye Res. 2016 Aug;149:100-6.

[2]. Inhibition of p38 MAPK Phosphorylation Is Critical for Bestatin to Enhance ATRA-Induced Cell Differentiation in Acute Promyelocytic Leukemia NB4 Cells. Am J Ther. 2016 May-Jun;23(3):e680-9.

[3]. The effects of bestatin on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunocompromised mice. Immunopharmacol Immunotoxicol. 2013 Feb;35(1):133-8.

[4]. Bestatin inhibits cell growth, cell division, and spore cell differentiation in Dictyostelium discoideum. Eukaryot Cell. 2012 Apr;11(4):545-57.

Ubenimide (also known as bestinatin) is a competitive protease inhibitor. It inhibits aminopeptidase B, leukotriene A4 hydrolase, and aminopeptidase N. Its potential for treating acute myeloid leukemia is currently under investigation. Ubenimide has been reported to be found in Streptomyces abikoensis and Streptomyces olivoreticuli, with relevant data available. Ubenimide is a microbial metabolite and dipeptide with potential immunomodulatory and antitumor activities. Ubenimide competitively inhibits multiple aminopeptidases, including aminopeptidase B, aminopeptidase N, and leucine aminopeptidase. Aminopeptidases are involved in cell adhesion and tumor cell invasion. Therefore, inhibition of aminopeptidases may be part of the reason for urbenimide's antitumor effect. The drug can also activate T lymphocytes, macrophages, and bone marrow stem cells, and stimulate the release of interleukin-1 and interleukin-2, thereby further enhancing its antitumor activity.

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Drug Indications
Used as adjuvant therapy for acute and chronic myeloid leukemia, lung cancer, and nasopharyngeal carcinoma. Also used to treat hypercholesterolemia.

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Bestatin (as bestatin methyl ester, BME) is a specific inhibitor of Zn²⁺-binding aminopeptidases. It inhibits cell proliferation and induces apoptosis in normal and cancer cells. Bestatin is currently used in the treatment of acute myeloid leukemia and lung squamous cell carcinoma. It may also have future uses in lung adenocarcinoma, esophageal adenocarcinoma, choriocarcinoma, and uterine cervical carcinoma. Bestatin also has anti-inflammatory properties, modulating cytokine and chemokine production by monocytes and macrophages.
In Dictyostelium discoideum, only two Zn²⁺-binding aminopeptidases have been identified: puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PsaA is differentially expressed throughout growth and development, and its expression is regulated by developmental morphogens (induced by cAMP, repressed by DIF-1 or NH₄Cl). Bestatin methyl ester specifically interacts with PsaA and inhibits its aminopeptidase activity.
Overexpression of PsaA-GFP (primarily nuclear) inhibits spore cell differentiation and drives cells to stalk cell pathway, while cytoplasmic PsaA (PsaAΔNLS2-GFP) drives cells to spore cell pathway. Bestatin treatment mimics the effect of PsaA overexpression on spore differentiation.
The study suggests that PsaA is functionally linked to Cdk5, and inhibition of PsaA by bestatin affects cell cycle progression and differentiation. [4]

Bestatin hydrochloride;65391-42-6;Bestatin trifluoroacetate;223763-80-2

72172

White to off-white solid powder

1.2±0.1 g/cm3

604.7±55.0 °C at 760 mmHg

245 °C (dec.)(lit.)

319.5±31.5 °C

0.0±1.8 mmHg at 25°C

1.557

2.64

4

5

8

22

367

3

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 : ~8.33 mg/mL (~27.01 mM)

Solubility in Formulation 1: ≥ 0.83 mg/mL (2.69 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (2.69 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 8.3 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: ≥ 0.83 mg/mL (2.69 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 8.3 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.)