Novobiocin targets multiple molecular targets. Its primary antibacterial target is DNA gyrase (bacterial topoisomerase II), specifically binding to the GyrB subunit and inhibiting ATP-dependent DNA supercoiling. This disrupts bacterial DNA replication and leads to cell death. Novobiocin also targets eukaryotic topoisomerase II. Additionally, it is a heat shock protein 90 (Hsp90) antagonist, binding at a second ATP-binding site in the C-terminal domain of Hsp90 and disrupting the interaction of p23 and Hsp70 co-chaperones with the Hsp90 complex. This Hsp90 inhibition contributes to its potential anticancer activity.

Novobiocin (1 mM) disrupts nucleotide binding by competitively inhibiting ATP binding to gyrase B. It also interferes with the co-chaperones' connection with Hsp90, namely Hsc70 and p23[1]. Novobiocin (200 µM; 24 h) reduces the clonogenic survival of human glioblastoma multiforme cells by inhibiting the rate of repair of both cis-DDP and BCNU-induced DNA interstrand cross-links[2]. Novobiocin (0.3 mM; 48 hours) causes an approximately three- to five-fold increase in apoptotic cells in K562, HL60, and Mutz-2 experiments that are dependent on the caspase-3/7 enzyme[5].
In vitro, Novobiocin demonstrates broad-spectrum Gram-positive antibiotic activity. It is an effective antistaphylococcal agent active against MRSA. It also shows activity against Staphylococcus epidermidis. As a DNA gyrase inhibitor, it disrupts bacterial DNA synthesis. As an Hsp90 antagonist, it shows potential for studying Hsp90-related biology. Detailed in vitro IC50 values for antibacterial activity are not extensively reported in the available literature.

Anti-infection activity of Novobiocin (25, 50, 100, and 200 mg/kg; sc; four times at 1, 5, 24, and 48 hours after infection) has been shown in mice infected with Streptococcus pneumoniae that is resistant to amoxicillin[3].
In vivo, Novobiocin is an orally active antibiotic. It has been used clinically to treat infections and has been studied for its potential in treating highly resistant pneumococcal infections. It also shows anti-orthopoxvirus activity. Detailed in vivo efficacy data from animal models are not extensively reported in the available literature.

Non-cell-based enzyme assays for Novobiocin use purified DNA gyrase enzyme. The compound is incubated with the enzyme, DNA substrate, and ATP at varying concentrations. DNA gyrase activity is measured by assessing DNA supercoiling using gel electrophoresis or fluorescence-based assays. IC50 values for enzyme inhibition are determined. Hsp90 binding assays may be performed using purified Hsp90 protein and labeled ATP or using surface plasmon resonance.

Cellular assays for Novobiocin utilize bacterial cultures to assess antibacterial activity. Minimum inhibitory concentrations (MICs) are determined using broth microdilution or agar dilution methods against various bacterial strains including MRSA and S. epidermidis. For Hsp90 inhibition studies, cancer cell lines are treated with the compound, and cell proliferation, apoptosis, and Hsp90 client protein levels are assessed.

Animal/Disease Models: 30 g adult female Swiss mice (sepsis induced by the penicillin-susceptible strain (AR33118))[3]
Doses: 25, 50, 100, 200 mg/kg
Route of Administration: Sc; given at 1, 5, 24 and 48 h after infection
Experimental Results: demonstrated anti-infection activity in mice infected with amoxicillin-resistant S. pneumoniae.
In vivo animal models for Novobiocin include standard infection models such as murine models of bacterial infection. The compound is administered orally or parenterally, and efficacy is assessed by survival, bacterial burden in target organs, and clinical signs of infection. For Hsp90 inhibition studies, xenograft models may be used. Detailed protocols are not extensively reported in the available literature.

Absorption, Distribution and Excretion
Oral bioavailability is negligible. In a phase I clinical trial, patients with refractory cancer received VP-16 on days 1, 3, and 5. Antiemetics, including ondansetron and dexamethasone, were administered 60 minutes before VP-16 administration. Neomycin was administered orally 30 minutes before VP-16 administration, with doses progressively increased in consecutive patient groups according to a standard dose escalation regimen. Treatment cycles were repeated every 4 weeks. Plasma neomycin concentrations were determined by high-performance liquid chromatography during the first treatment cycle. A total of 33 patients received 69 treatment cycles. Eleven patients received an initial dose of VP-16 of 120 mg/m², of whom 3 experienced neutropenic fever. The VP-16 dose was reduced to 100 mg/m², and an additional 22 patients were enrolled. The dose range of novimycin is 3 to 9 g. When the dose of novimycin was at least 5.5 g, the plasma concentration was maintained at at least 150 μM for 24 hours.

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Biological half-life
6 hours
Novimycin was administered orally for 96 hours; cyclophosphamide 750 mg/m² was administered intravenously 48 hours later. 34 patients received 65 treatment cycles. …Of the 19 patients who received ≥ 4 g daily, 18 achieved serum concentrations ≥ 100 μg/ml at steady state, consistent with concentrations observed in in vitro and in vivo experiments. The half-life of novimycin in mice was 82 minutes, significantly shorter than the human half-life (6.0 hours).
Pharmacokinetic properties of Novobiocin include a molecular weight of 612.62 g/mol and molecular formula C31H36N2O11. CAS number is 303-81-1. The compound is orally active. It is soluble in DMSO at 100 mg/mL (163.23 mM). Purity is not specified but is of research grade. Storage conditions: -20°C, sealed storage, away from moisture. Detailed PK parameters such as half-life and bioavailability are not extensively reported.

Protein Binding
95%
Novobiocin is an antibiotic with a well-established safety profile from clinical use. It is effective against MRSA and other Gram-positive bacteria. Detailed toxicological data are not extensively reported in the available literature. As an antibiotic, potential side effects include gastrointestinal disturbances and allergic reactions. The compound is for research use only and not for human consumption in a research context.

[1]. The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone. J Biol Chem. 2000 Nov 24;275(47):37181-6.

[2]. Topoisomerase II inhibition and altered kinetics of formation and repair of nitrosourea and cisplatin-induced DNA interstrand cross-links and cytotoxicity in human glioblastoma cells. Cancer Res. 1993 Dec 1;53(23):5663-8.

[3]. Comparative efficacy of novobiocin and amoxicillin in experimental sepsis caused by beta-lactam-susceptible and highly resistant pneumococci. Int J Antimicrob Agents. 2010 Jun;35(6):544-9.

[4]. A phase I clinical trial of novobiocin, a modulator of alkylating agent cytotoxicity. Cancer Res. 1991 Jan 15;51(2):510-3.

[5]. Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response.Blood. 2018 Jul 19;132(3):307-320.

[6]. Smee DF. Progress in the discovery of compounds inhibiting orthopoxviruses in animal models. Antivir Chem Chemother. 2008;19(3):115-24.

Novobiocin is a coumarin antibiotic extracted from Streptomyces niveus. It has dual functions: antibacterial activity, inhibition of DNA topoisomerase (ATP hydrolase) activity, as a metabolite of Escherichia coli, and hepatoprotective agent. It is a hexoside, monocarboxylic acid amide, monosaccharide derivative, hydroxycoumarin, ether, carbamate, and phenolic compound. It is the conjugate acid of novimycin (1-). Novobiocin is an antibiotic compound derived from Streptomyces niveus. Its chemical structure is similar to that of coumarin. Novobiocin binds to DNA gyrase, inhibiting the activity of adenosine triphosphate (ATPase). (Excerpt from Reynolds, Martindale Pharmacopoeia, 30th edition, p. 189) Novobiocin sodium is the salt form of novimycin, initially approved in September 1964 for the treatment of serious infections caused by susceptible Staphylococcus aureus strains, especially when other less toxic antibiotics are unavailable. In 2009, the U.S. Food and Drug Administration (FDA) halted the sale of novimycin sodium due to safety and efficacy concerns. Novimycin is reportedly found in Streptomyces, Streptomyces leucopterus, and other microorganisms for which relevant data are available. Novimycin is an aminocoumarin antibiotic produced by the actinomycete Streptomyces niveus and possesses antibacterial activity. Like other aminocoumarin antibiotics, novimycin inhibits bacterial DNA synthesis by targeting bacterial DNA gyrase and its associated enzyme, DNA topoisomerase IV. This antibiotic has been used to treat Gram-positive bacterial infections. Novimycin is an antibiotic compound derived from Streptomyces niveus. Its chemical structure is similar to that of coumarins. Novimycin binds to DNA gyrase and blocks the activity of adenosine triphosphatase (ATPase). (Excerpt from Reynolds Martindale Pharmacopoeia, 30th edition, p. 189) See also: Novimycin sodium (salt form); Novimycin calcium (salt form). Indications Novimomycin is used to treat infections caused by Staphylococcus and other susceptible bacteria. Mechanism of Action Novimomycin is an aminocoumarin that acts by inhibiting the GyrB subunit of bacterial DNA gyrase, an enzyme involved in energy transduction. Similar to other aminocoumarin antibiotics, it acts as a competitive inhibitor of the GyrB-catalyzed ATPase reaction.
Novobiocin is also known as Albamycin and Cathomycin. It has CAS number 303-81-1. It is an aminocoumarin antibiotic produced by Streptomyces niveus. It is a DNA gyrase inhibitor that binds to the GyrB subunit. It is also an Hsp90 antagonist. It exhibits broad-spectrum Gram-positive antibiotic activity and has been used clinically for MRSA. It also shows anti-orthopoxvirus activity. It is used for research purposes.

C31H36N2O11

612.63

612.231

303-81-1

Novobiocin sodium;1476-53-5

54675769

White to off-white solid powder

1.4±0.1 g/cm3

848.2±65.0 °C at 760 mmHg

170-172°C (lit.)

466.8±34.3 °C

0.0±3.3 mmHg at 25°C

1.640

2.37

5

11

9

44

1150

4

CC1=C(C=CC2=C1OC(=O)C(=C2O)NC(=O)C3=CC(=C(C=C3)O)CC=C(C)C)O[C@H]4[C@@H]([C@@H]([C@H](C(O4)(C)C)OC)OC(=O)N)O

YJQPYGGHQPGBLI-KGSXXDOSSA-N

InChI=1S/C31H36N2O11/c1-14(2)7-8-16-13-17(9-11-19(16)34)27(37)33-21-22(35)18-10-12-20(15(3)24(18)42-28(21)38)41-29-23(36)25(43-30(32)39)26(40-6)31(4,5)44-29/h7,9-13,23,25-26,29,34-36H,8H2,1-6H3,(H2,32,39)(H,33,37)/t23-,25+,26-,29-/m1/s1

[(3R,4S,5R,6R)-5-hydroxy-6-[4-hydroxy-3-[[4-hydroxy-3-(3-methylbut-2-enyl)benzoyl]amino]-8-methyl-2-oxochromen-7-yl]oxy-3-methoxy-2,2-dimethyloxan-4-yl] carbamate

U-6391 U 6391 NovobiocinCathomycin,U6591, U-6591, Inabiocin, Albadry, Streptonivicin, Albamycin

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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