Trehalase, tyrosinase. Validamycin A inhibits trehalase from Rhizoctonia solani with an IC₅₀ of 7.2 × 10⁻⁵ M (72 μM), and its aglycone validoxylamine A inhibits trehalase competitively with a Kᵢ of 1.9 × 10⁻⁹ M (1.9 nM). It inhibits mushroom tyrosinase with an IC₅₀ of 19.23 ± 0.26 mM, and a mixed-type inhibition constant Kᵢ of 5.893 ± 0.038 mM. [1,2,3]
Antibiotic[1], IC50: 72 μM (trehalase)[2], Ki: 5.893 mM (tyrosinase)[3]

Trehalase inhibition in Rhizoctonia solani: Validamycin A showed potent inhibitory activity against trehalase of R. solani, with an IC₅₀ of 7.2 × 10⁻⁵ M. Validoxylamine A, the aglycone, exhibited even greater inhibitory activity with a Kᵢ of 1.9 × 10⁻⁹ M. Validamycin A showed no significant inhibition against cellulase, pectinase, chitinase, α-amylase, α-glucosidase, or β-glucosidase from R. solani. [2]
Tyrosinase inhibition: Validamycin A inhibited mushroom tyrosinase in a dose-dependent, reversible manner with an IC₅₀ of 19.23 ± 0.26 mM. The inhibition was mixed-type, with a Kᵢ of 5.893 ± 0.038 mM and an α value of 2.096 ± 0.546. The inactivation kinetics followed first-order kinetics with two phases (fast and slow), with rate constants ranging from 1.54 × 10⁻³ to 3.60 × 10⁻³ s⁻¹ for the fast phase and 0.45 × 10⁻³ to 1.45 × 10⁻³ s⁻¹ for the slow phase. [3]
Effect on fungal growth and morphology: Validamycin A at concentrations as low as 0.01 μg/mL caused abnormal branching of hyphae in R. solani in the "dendroid-test method". It did not significantly suppress growth on nutritionally rich medium but specifically induced abnormal branching and cessation of colony development on water-agar. [2]
Effect on Aspergillus flavus: Validamycin A at 1 μg/mL significantly increased trehalose levels in A. flavus conidia, delayed conidial germination (particularly at 10 and 12 hours), and decreased fungal adherence. The minimal inhibitory concentration (MIC) of validamycin A against A. flavus ATCC204304 was 1 μg/mL. [1]
Combination effect with amphotericin B: In checkerboard assays, validamycin A and amphotericin B showed an additive effect on A. flavus ATCC204304 (FICI = 0.625). In amphotericin B-resistant clinical isolates of A. flavus (MIC > 4 μg/mL), the combination exhibited a synergistic effect with FICI values of 0.25–0.28, with validamycin A and amphotericin B concentrations at 0.125 μg/mL and 2 μg/mL, respectively. [1]
Effect on trehalose metabolism in R. solani: Validamycin A at 0.1 μg/mL significantly suppressed the degradation of intracellular trehalose in R. solani mycelia, indicating inhibition of trehalase activity in vivo. [2]
Cellular uptake and metabolism in R. solani: Validamycin A was taken up by R. solani mycelia and hydrolyzed intracellularly by β-glucosidase to yield validoxylamine A, the active trehalase inhibitor. Validamycin A (β-D-glucoside) was more readily taken up than its aglycone (validoxylamine A) or the α-D-glucoside (validamycin D). [2]
Validamycin A (0.5–1 μg/mL; 18 hours) stops A from growing. conidial germination and inhibits flavus [1]. For human bronchial epithelial cells, validamycin A is not cytotoxic [1]. HIS85, HIS244, GLU256, HIS259, and ASN260 are among the residues in the tyrosinase active site that Validamycin A directly binds to [1].

Trehalase inhibition assay (R. solani): Trehalase activity was measured by colorimetric determination of D-glucose released from trehalose. The reaction mixture contained 125 μL enzyme solution, 50 μL of 0.4 M trehalose, 200 μL inhibitor solution or water, and 125 μL of 0.2 M phosphate buffer (pH 6.0). After 15 minutes at 37°C, released glucose was measured by the glucose oxidase method. [2]
Tyrosinase inhibition assay: Tyrosinase activity was measured spectrophotometrically by monitoring the increase in absorbance at 475 nm due to dopachrome formation from L-DOPA. The assay mixture contained 2 mM L-DOPA, 1.0 μg/mL tyrosinase, and varying concentrations of validamycin A in 50 mM sodium phosphate buffer (pH 7.0) at 25°C. [3]
Inhibition kinetics analysis: Lineweaver-Burk double reciprocal plots were used to determine the inhibition type. Secondary plots of slope and y-intercept versus inhibitor concentration were linearly fitted to calculate Kᵢ and α values using equations for mixed-type inhibition. [3]
Inactivation kinetics: Time-course studies of tyrosinase inhibition were performed by incubating the enzyme with varying concentrations of validamycin A (3.125–50 mM). Aliquots were collected at different time intervals, and the residual activity was measured. Semi-logarithmic plots revealed biphasic first-order inactivation kinetics, with rate constants determined from the slopes. [3]
Fluorescence spectroscopy: Intrinsic tryptophan fluorescence of tyrosinase was measured after excitation at 280 nm (emission 300–400 nm) to monitor tertiary structure changes upon validamycin A binding. ANS-binding fluorescence (excitation 390 nm, emission 400–600 nm) was used to assess changes in surface hydrophobicity. The binding constant (K = 0.06 ± 0.012 mM⁻¹) and binding number (n = 1.06 ± 0.37) were calculated from fluorescence quenching data. [3]
Computational docking and molecular dynamics: The binding mode between validamycin A and tyrosinase was investigated using AutoDock Vina for docking and CHARMM with CGENFF parameters for molecular dynamics simulations. The lowest-energy, largest, and active-site clusters were selected for 10 ns simulations. Five residues (HIS85, HIS244, GLU256, HIS259, and ASN260) were identified as interacting with validamycin A, with distances less than 4 Å. [3]

Aspergillus flavus viability assay (XTT): A. flavus conidia (10³ cells) were incubated in various culture media with or without validamycin A in 96-well plates at 37°C for 18 hours. XTT solution (0.5 mg/mL in PBS) was added, incubated for 15 minutes, centrifuged, and the supernatant measured at 490 nm to assess fungal viability. [1]
Fungal adherence assay (crystal violet): A. flavus conidia (10⁵ cells/mL) were incubated in Sabouraud dextrose broth in 96-well plates at 37°C for 24 hours with or without validamycin A (1 μg/mL). After gentle washing, cells were stained with 0.1% crystal violet, destained with ethanol, and absorbance was measured at 600 nm. [1]
Trehalose measurement in A. flavus conidia: A. flavus conidia (2 × 10⁸ cells) were collected after 5 days of growth on SDA with or without validamycin A (1 μg/mL). Conidia were boiled, centrifuged, and trehalose in the supernatant was measured using the glucose oxidase assay. [1]
Germination assay: A. flavus conidia (1 × 10⁸ cells) were incubated in Sabouraud dextrose broth at 37°C with shaking (200 rpm) with or without validamycin A (1 μg/mL). At various time points, 100 conidia were counted microscopically to determine the percentage of germinated conidia. [1]
Cytotoxicity assay (LDH): BEAS-2B human bronchial epithelial cells (1 × 10⁴ cells) were incubated with varying concentrations of validamycin A (1 μg/mL to 1 mg/mL) for 24 hours. LDH release was measured at 450 nm, and cytotoxicity percentage was calculated relative to high and low controls. No significant cytotoxicity was observed at concentrations up to 1 mg/mL. [1]
Cell Proliferation Assay[1]
Cell Types: Aspergillus flavus ATCC204304
Tested Concentrations: 0.5 μg/mL, 1 μg/mL
Incubation Duration: 18 hrs (hours)
Experimental Results: Inhibited the growth of Aspergillus flavus.

Metabolism / Metabolites
In animals, it is broken down into glucose and amine residues. Biological Half-Life Less than or equal to 5 hours.

Toxicity Data
LC50 (Rat) > 5,000 mg/m3

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Non-human Toxicity Values LD50 (Rats, Oral) > 20,000 mg/kg
LD50 (Mouse, Oral) > 20,000 mg/kg
LD50 (Rats, Dermal) > 5,000 mg/kg

[1]. The Inhibitory Effect of Validamycin A on Aspergillus flavus. Int J Microbiol. 2020: 3972415.

[2]. Effect of validamycins on glycohydrolases of Rhizoctonia solani. J Antibiot (Tokyo). 1987 Apr;40(4):526-32.

[3]. The effect of validamycin A on tyrosinase: inhibition kinetics and computational simulation. Int J Biol Macromol. 2013 Apr;55:15-23.

Validamycin A belongs to the validamycin class of compounds. Its structure is (1R,2S,3S,4S,6R)-4-amino-6-(hydroxymethyl)cyclohexane-1,2,3-triol, where the hydroxyl group at position 1 is converted to β-D-glucoside, and a hydrogen atom bonded to the nitrogen atom is replaced by (1R,4R,5R,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohexane-2-en-1-yl. It is the main validamycin produced by Streptomyces hygroscopicus. It is an EC 2.4.1.231 [α,α-trehalose phosphorylase (configuration-maintaining form)] inhibitor, an EC 2.4.1.64 (α,α-trehalose phosphorylase) inhibitor, an EC 3.2.1.28 (α,α-trehalase) inhibitor, and an antifungal pesticide. It belongs to the class of potentiin compounds, and is a secondary amino compound, polyol, and antibiotic bactericide. It is the conjugate base of potentiin A(1+). Pulpitin A has been reported to exist in Streptomyces anthocyanicus, Streptomyces hygroscopicus, and Streptomyces lividans, and relevant data are available.

C20H35NO13

497.49

497.21

C, 48.29; H, 7.09; N, 2.82; O, 41.81

37248-47-8

443629

White to off-white solid powder

1.7±0.1 g/cm3

813.7±65.0 °C at 760 mmHg

130-135ºC

445.9±34.3 °C

0.0±6.6 mmHg at 25°C

1.689

-5.43

12

14

7

34

697

14

C1[C@@H]([C@H]([C@@H]([C@H]([C@H]1N[C@H]2C=C([C@H]([C@@H]([C@H]2O)O)O)CO)O)O)O[C@H]3[C@@H]([C@H]([C@@H]([C@H](O3)CO)O)O)O)CO

JARYYMUOCXVXNK-CSLFJTBJSA-N

InChI=1S/C20H35NO13/c22-3-6-1-8(12(26)15(29)11(6)25)21-9-2-7(4-23)19(17(31)13(9)27)34-20-18(32)16(30)14(28)10(5-24)33-20/h1,7-32H,2-5H2/t7-,8+,9+,10-,11-,12+,13+,14-,15+,16+,17-,18-,19-,20+/m1/s1

(2R,3R,4S,5S,6R)-2-[(1R,2R,3S,4S,6R)-2,3-dihydroxy-6-(hydroxymethyl)-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-en-1-yl]amino]cyclohexyl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol

Validamycin; jinggangmycin; VALIDAMYCIN A; 37248-47-8; Valimon;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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)

H2O : 125 mg/mL (251.26 mM)

Solubility in Formulation 1: 100 mg/mL (201.01 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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