Aminoglycoside
Hygromycin B binds to helix 44 of 16S rRNA in the small ribosomal subunit, specifically interacting with nucleotides C1403, G1405, G1494, U1495, C1496, and U1498 (E. coli numbering). [3]

Streptomyces hygroscopicus synthesizes hygromycin B, an aminocyclitol antibiotic that strongly inhibits both 70S and 80S ribosomes[1]. At a concentration of 0.38 mM, hygromycin B completely stops the growth of yeast cells in rich media, most likely by stopping cytoplasmic ribosomes from synthesizing proteins. Low concentrations of hygromycin B significantly inhibit the synthesis of polypeptides in cell-free extracts from yeast, wheat germ, and rabbit reticulocytes. The antibiotic stops the translocation of elongation factor EF-2, which inhibits yeast polysomes from elongating peptide chains. Hygromycin B's suppression of translocation may be due to the stabilization of peptidyl-tRNA attached to the ribosomal acceptor site[2].

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Hygromycin B inhibits polyphenylalanine synthesis in cell-free systems. In a system from Streptomyces lividans, the drug concentration causing 50% inhibition was approximately 0.5 μM; in a system from the producing organism Streptomyces hygroscopicus, 136 μM was required for 50% inhibition (indicating that ribosomes from S. hygroscopicus are sensitive to the drug, but the presence of a modifying enzyme in the S100 fraction confers resistance). [1]
Hygromycin B induces misreading both in vivo and in vitro, thereby promoting phenotypic suppression. [1]
Hygromycin B is inactive against protein synthesis after enzymatic phosphorylation. The phosphorylated product, 7″-O-phosphoryl-hygromycin B, completely lacks biological activity in vitro (no inhibition of polyphenylalanine synthesis in S. lividans cell-free system at concentrations up to 30 μM). Dephosphorylation with alkaline phosphatase fully restores its inhibitory activity. [1]

By preventing ribosome translocation, hygromycin B inhibits protein synthesis without significantly altering ribosome positioning in vivo[3].
Hygromycin B resistance is conferred in vivo in transgenic mice through constitutive expression of the bacterial hygR gene[4].

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In Mycobacterium smegmatis, spontaneous hygromycin B-resistant mutants were isolated on medium containing 50 μg/mL hygromycin B. Sequencing of the 16S rRNA gene identified point mutations at positions U1406C (24 of 35 mutants), C1496U (6 of 35), and U1498C (5 of 35). [3]
- The MICs for wild-type M. smegmatis were 0.03 μg/mL (33.4 μM). Mutants U1406C showed MIC of 1024 μg/mL (1.96 mM); C1496U showed MIC of 512 μg/mL (0.97 mM); U1498C showed MIC of 128–256 μg/mL (0.24–0.48 mM). [3]
- The generation times of U1406C and C1496U mutants were significantly increased (5.3 ± 0.8 h and 5.2 ± 1.1 h, respectively) compared to wild-type (3.4 ± 0.4 h). U1498C showed a generation time of 3.8 ± 0.3 h. [3]
- Attempts to isolate resistant mutants from parental M. smegmatis SMR5 (carrying two functional rRNA operons) were unsuccessful (mutation rate < 10⁻¹⁰), indicating that the resistance mutations act in a recessive manner. [3]
- Using a RecA-mediated homologous recombination assay, plasmids carrying the resistance mutations (U1406C, C1496U, U1498C) on a nonfunctional rRNA fragment were introduced into M. smegmatis rn⁻. Hygromycin B-resistant recombinants were obtained, confirming that these mutations are causative for resistance. [3]

Hygromycin B is an aminoglycoside antibiotic active against prokaryotic and eukaryotic ribosomes. Ribosomal alterations in bacteria conferring resistance to hygromycin B have not been described, prompting us to use a single rRNA allelic derivative of the gram-positive bacterium Mycobacterium smegmatis for investigation of the molecular mechanisms involved in ribosomal resistance to hygromycin B in eubacteria. Resistance mutations were found to localize exclusively in 16S rRNA. The mutations observed, i.e., 16S rRNA U1406C, C1496U, and U1498C (E. coli numbering), are in close proximity to the hygromycin B binding site located in conserved helix 44 of 16S rRNA. The 16S rRNA positions involved in hygromycin B resistance are highly conserved in all three domains of life, explaining the lack of specificity and general toxicity of hygromycin B[3].

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Hygromycin B, an aminocyclitol antibiotic that strongly inhibits both 70S and 80S ribosomes, is synthesized by Streptomyces hygroscopicus. Ribosomes from this Gram-positive mycelial bacterium are inhibited in vitro by the antibiotic. In contrast, the streptomycete is highly resistant to the drug in vivo since it possesses hygromycin B phosphotransferase activity. This enzyme has been shown by gel filtration to have a molecular weight of 42000, and to modify its antibiotic substrate to produce 7"-O-phosphoryl-hygromycin B which totally lacks biological activity both in vivo and in vitro[1].

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Hygromycin B phosphotransferase (HPH) activity assay: The radiochemical method uses [γ-³²P]ATP as the phosphate donor. Reaction mixtures (50 μL) contain 10 μL buffer I (100 mM Tris/MES, pH 7.5, 50 mM MgCl₂, 500 mM NH₄Cl, 2.5 mM dithiothreitol), 10 μL enzyme preparation, 1 μg hygromycin B, and 10 μL 1.5 mM [γ-³²P]ATP (15 mCi/mmol). Assays are performed for 20 min at 30°C, stopped by heating at 75°C, and an aliquot is applied to phosphocellulose paper (Whatman P-81), washed with tap water and distilled water, and radioactivity is counted. Blanks are run without hygromycin B. [1]
Spectrophotometric assay: Used for continuous measurement of reaction velocities, with 0.8 U HPH, 2.5-100 μM hygromycin B, and 30-3000 μM ATP. [1]
Kinetic constants: The Kₘ of HPH for hygromycin B was approximately 2.35 μM (by spectrophotometric assay) or 1.5 μM (by radiochemical assay); the Kₘ for ATP was 57 μM (spectrophotometric) or 76 μM (radiochemical). Substrate inhibition was observed at hygromycin B concentrations above 5 μM. [1]
The site of phosphorylation on hygromycin B was determined by ¹H NMR to be the 7″-hydroxyl group of the destomic acid moiety. [1]

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B[2].

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MICs were determined in microtiter plates. Starting cultures contained bacterial cells at OD₆₀₀ = 0.025, and hygromycin B was added in twofold dilutions. MIC was defined as the drug concentration at which growth was completely inhibited after 72 h of incubation at 37°C (approximately 24 generations). [3]
- Growth rates were determined by measuring generation times in liquid culture. [3]
- Spontaneous resistant mutants were selected on LB agar containing 50 μg/mL hygromycin B. [3]

Dissolve hygromycin B in sterile water. Hygromycin B i.p. is administered as a single dose to the hygR-carrying C57BL/6J-TgN(pPWL512hyg)1Em mice at a starting dose of 2.7 mg/kg and increasing by 50% with each subsequent dose. The same volume of sterile saline is administered to wild-type C57BL/6J control mice. There is 0.5 mL injected in total. For ten days in a row, the animals' body weight and general health are observed every day[4].

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Hygromycin B inhibits protein synthesis in archaebacteria, eubacteria, fungi, and higher eukaryotes, indicating universal toxicity. [3]
- The nucleotides conferring resistance (U1406, C1496, U1498) are universally conserved across all domains of life, explaining the broad-spectrum toxicity and lack of selectivity. [3]

[1]. Biochemical basis of resistance to hygromycin B in Streptomyces hygroscopicus--the producing organism. J Gen Microbiol. 1985 Jun;131(6):1289-98.

[2]. Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochim Biophys Acta. 1978 Dec 21;521(2):459-69.

[3]. Role of 16S rRNA Helix 44 in Ribosomal Resistance to Hygromycin B. Antimicrob Agents Chemother. 2003 May;47(5):1496-502.

Amorphous solid or brownish powder. (NTP, 1992)
Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus. It kills bacteria, fungi, and higher eukaryotic cells by inhibiting protein synthesis.
An aminoglycoside antibiotic produced by Streptomyces hygroscopicus. It is used as an anthelmintic to treat infections of large roundworms, nodular nematodes, and whipworms in pigs.
See also: Hygromycin B (note moved to).

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Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus. It inhibits protein synthesis by blocking ribosomal translocation, without causing significant misreading in vivo. [3]
- In contrast to classical 2-deoxystreptamine aminoglycosides, hygromycin B has a unique structure featuring a 5-substituted N-methyl-2-deoxystreptamine with a dual ether linkage between two sugar moieties, forming a fourth, five-membered ring. [3]
- The drug binds in a sequence-specific manner near the top of helix 44 of 16S rRNA, between the A and P sites, and interacts with nucleotides C1403, G1405, G1494, U1495, C1496, and U1498. [3]
- This study is the first to report ribosomal resistance mutations to hygromycin B in eubacteria, localizing to 16S rRNA positions U1406, C1496, and U1498. [3]
- The recessive nature of these mutations explains why resistant mutants are difficult to isolate in bacteria with multiple rRNA operons. [3]
- The universal conservation of the resistance-associated nucleotides across prokaryotes and eukaryotes accounts for the lack of species specificity and general toxicity of hygromycin B. [3]

C20H37N3O13

527.52008

527.232

C, 45.54; H, 7.07; N, 7.97; O, 39.43

31282-04-9

20054942

White to light yellow solid powder

1.7±0.1 g/cm3

897.6±65.0 °C at 760 mmHg

160-180ºC

496.7±34.3 °C

0.0±0.6 mmHg at 25°C

1.672

0.64

11

16

6

36

756

14

CN[C@H]1C[C@H]([C@@H]([C@H]([C@@H]1O)O[C@H]2[C@@H]3[C@H]([C@H]([C@H](O2)CO)O)OC4(O3)[C@@H]([C@H]([C@H]([C@H](O4)C(CO)N)O)O)O)O)N

GRRNUXAQVGOGFE-HUCHGKBZSA-N

InChI=1S/C20H37N3O13/c1-23-7-2-5(21)9(26)15(10(7)27)33-19-17-16(11(28)8(4-25)32-19)35-20(36-17)18(31)13(30)12(29)14(34-20)6(22)3-24/h5-19,23-31H,2-4,21-22H2,1H3/t5-,6+,7+,8-,9+,10-,11+,12-,13+,14-,15-,16+,17+,18-,19+,20?/m1/s1

D-Streptamine, O-6-amino-6-deoxy-L-glycero-D-galacto-heptopyranosylidene(1-2-3)-O-beta-D-talopyranosyl-(1->)-2-deoxy-N3-methyl-

HygromycinB, Hygrovetine, Hygromix 2.4, Antihelmycin, Hygromix-8;Hygromycin-B; AI3-29796; hygromycin b; 31282-04-9; Hygrovetine; MFCD06795479; (2S,3R,3A'S,4S,4'S,5R,6R,6'R,7'S,7a'S)-4'-(((1R,2S,3R,5S,6R)-3-amino-2,6-dihydroxy-5-(methylamino)cyclohexyl)oxy)-6-((R)-1-amino-2-hydroxyethyl)-6'-(hydroxymethyl)octahydro-4'H-spiro[pyran-2,2'-[1,3]dioxolo[4,5-c]pyran]-3,4,5,7'-tetraol; 1217468-11-5; C20H37N3O13; CHEMBL4647156; AI3 29796; AI329796

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~100 mg/mL (~189.57 mM )
H2O : 50~100 mg/mL (~94.78 mM )

Solubility in Formulation 1: ≥ 5 mg/mL (9.48 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 50.0 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: ≥ 5 mg/mL (9.48 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 50.0 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: ≥ 5 mg/mL (9.48 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.74 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (4.74 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: ≥ 0.5 mg/mL (0.95 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 7: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 5 mg/mL (9.48 mM)

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Solubility in Formulation 8: 100 mg/mL (189.57 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.)