Topoisomerase II; Topoisomerase IV
Bacterial DNA gyrase (MIC = 0.25-1 μg/mL against Staphylococcus aureus) [2][5]
Bacterial topoisomerase IV (MIC = 0.5-2 μg/mL against Staphylococcus aureus) [2]
TAR RNA-binding protein 2 (TRBP) [1][3]

In vitro activity: Enoxacin (AT 2266) has no effect on cells expressing GFP alone, but it increases siGFP-mediated gene knockdown mediated by siRNA against EGFP in HEK293 cells-based reporter system in a dose-dependent manner, with a median effective concentration (EC50) of ~30 µM. In HEK293 cells, enoxacin (50 µM) facilitates siRNA duplex loading onto RISCs and miRNA processing[3].
Enoxacin has no impact on the way that Dicer alone processes pre-let-7 or pre-miR-30a. On the other hand, the combination of Enoxacin and TRBP can improve let-7 or pre-miR-30a processing[3].
Enoxacin and TRBP can improve let-7 or pre-miR-30a processing[3]. 90% of Escherichia coli, Aeromonas, Enterobacter, Serratia, Proteus mirabilis, and Morganella morganii are inhibited by enoxacin at concentrations of less than or equal to 0.8 micrograms/ml[5].

---

Against Staphylococcus aureus strains, Enoxacin (AT-2266) exhibited potent antibacterial activity, with MIC values of 0.25-1 μg/mL (targeting DNA gyrase) and 0.5-2 μg/mL (targeting topoisomerase IV). It inhibited bacterial DNA replication and transcription by interfering with these two enzymes [2][5]
- Against various Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae) and some Gram-positive bacteria, Enoxacin (AT-2266) showed antibacterial activity, with MIC values ranging from 0.125 to 8 μg/mL, superior to some beta-lactams and aminoglycosides against certain strains [5]
- In cancer cell lines (HeLa, A549, MCF-7), Enoxacin (AT-2266) acted as a cancer-specific growth inhibitor, inhibiting cell proliferation in a concentration-dependent manner. This effect was dependent on TRBP, as knockdown of TRBP abolished the antiproliferative activity [1]
- Enoxacin (AT-2266) enhanced TRBP-mediated microRNA (miRNA) processing, increasing the levels of mature miRNAs (e.g., miR-16, miR-let-7a) in cancer cells. It also promoted RNA interference (RNAi) efficiency by strengthening the interaction between TRBP and Dicer [1][3]

Enoxacin (AT 2266; 100 µM; 2 µl; injected into ear once a day for 3 consecutive days (days 12, 13 and 14)) increases Lv-siGFP'sabilityto knock down GFP mRNA (from 80% to 60%; 40% of GFP mRNA level remained); however, in the GFP transgenic line C57BL/6-Tg(ACTB-EGFP)1Osb/J (10 d old) using lentivirus expressing shGFP (Lv-siGFP; injected into ear for 10 days) alone has no effect on GFP expression[3].

Bacterial DNA gyrase activity assay: Purified Staphylococcus aureus DNA gyrase was incubated with supercoiled plasmid DNA in reaction buffer at 37°C. Enoxacin (AT-2266) was added at serial concentrations (0.0625-4 μg/mL), and the mixture was incubated for 60 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 55°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The inhibition of DNA gyrase-mediated supercoiling relaxation was quantified by measuring the intensity of supercoiled DNA bands [2]
- Bacterial topoisomerase IV activity assay: Isolated Staphylococcus aureus topoisomerase IV was incubated with relaxed plasmid DNA in reaction buffer. Enoxacin (AT-2266) was added at concentrations of 0.125-8 μg/mL, and the mixture was incubated at 37°C for 45 minutes. The reaction was stopped by adding stop solution, and DNA products were analyzed by agarose gel electrophoresis to assess the inhibition of DNA decatenation [2]

Antibacterial activity assay: Bacterial strains (Staphylococcus aureus, Escherichia coli) were cultured in Mueller-Hinton broth at 37°C with shaking. Enoxacin (AT-2266) was added at serial concentrations (0.03125-16 μg/mL), and bacterial growth was monitored by measuring optical density at 600 nm (OD600) after 24 hours. The MIC was defined as the lowest concentration inhibiting ≥90% bacterial growth [2][5]
- Cancer cell antiproliferation assay: Cancer cells (HeLa, A549) were seeded in 96-well plates at 3×10³ cells/well and treated with Enoxacin (AT-2266) at concentrations of 10-100 μM for 72 hours. Cell viability was measured using a tetrazolium-based colorimetric assay. TRBP knockdown experiments were performed via siRNA transfection to confirm target dependency [1]
- miRNA processing and RNAi assay: HeLa cells were transfected with miRNA expression plasmids or siRNA, then treated with Enoxacin (AT-2266) at 25-50 μM for 24-48 hours. Mature miRNA levels were detected by quantitative PCR. RNAi efficiency was evaluated by measuring the silencing of a reporter gene (e.g., luciferase) [1][3]
- TRBP-Dicer interaction assay: Cells were treated with Enoxacin (AT-2266) at 50 μM for 24 hours, then lysed and subjected to co-immunoprecipitation using anti-TRBP antibody. The precipitated proteins were analyzed by western blot to detect Dicer, assessing the enhancement of their interaction [3]

Absorption, Distribution and Excretion
Enoxacin is rapidly absorbed after oral administration, with an absolute oral bioavailability of approximately 90%. Metabolism/Metabolites Metabolized in the liver. Enoxacin inhibits certain isoenzymes of the hepatic microsomal cytochrome P-450 enzyme system. Following a single dose, over 40% of the drug is excreted unchanged in the urine within 48 hours. Biological Half-Life The plasma half-life is 3 to 6 hours.

Medication Use During Pregnancy and Lactation ◉ Overview of Medication Use During Lactation
Traditionally, fluoroquinolones are not recommended for use in infants due to concerns about adverse effects on the developing joints of infants. However, recent studies suggest the risk is minimal. Calcium in breast milk may prevent the absorption of small amounts of fluoroquinolones in breast milk, but there is currently insufficient data to confirm or refute this claim. Enoxacin may be acceptable for breastfeeding women, but close monitoring of the infant's gut microbiota is necessary, for example, for changes in diarrhea or candidiasis (thrush, diaper rash). However, alternative medications with available safety information are preferred.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

---

Protein Binding In healthy subjects, enoxacin binds to plasma proteins at a rate of approximately 40%; in patients with impaired renal function, the binding rate is approximately 14%.

[1]. Small molecule enoxacin is a cancer-specific growth inhibitor that acts by enhancing TAR RNA-binding protein 2-mediated microRNA processing. Proc Natl Acad Sci U S A. 2011 Mar 15;108(11):4394-9.

[2]. Target preference of 15 quinolones against Staphylococcus aureus, based on antibacterial activities and target inhibition. Antimicrob Agents Chemother. 2001 Dec;45(12):3544-7.

[3]. A small molecule enhances RNA interference and promotes microRNA processing. Nat Biotechnol. 2008 Aug;26(8):933-40.

[4]. Small molecules with big roles in microRNA chemical biology and microRNA-targeted therapeutics. RNA Biol. 2019 Jun;16(6):707-718.

[5]. In vitro activity of enoxacin, a quinolone carboxylic acid, compared with those of norfloxacin, new beta-lactams, aminoglycosides, and trimethoprim. Antimicrobial agents and chemotherapy, 1983. 24(5): p. 754-763.

Enoxacin is a 1,8-naphthidine derivative with the structure 1,4-dihydro-1,8-naphthidine, with an ethyl group at position 1, a carboxyl group at position 3, an oxygen substituent at position 4, a fluorine substituent at position 5, and a piperazine-1-yl group at position 7. It is an antibacterial drug used to treat urinary tract infections and gonorrhea. Enoxacin has antibacterial and DNA synthesis-inhibiting effects. It is a monocarboxylic acid, amino acid, 1,8-naphthidine derivative, N-arylpiperazine, quinolone antibiotic, and fluoroquinolone antibiotic.
A broad-spectrum 6-fluoronaphthidine ketone antibacterial agent (fluoroquinolone), its structure is related to nalidixic acid.
A broad-spectrum 6-fluoronaphthidine ketone antibacterial agent, its structure is related to nalidixic acid.
See also: Enoxacin sesquihydrate (its active moiety).

---

Indications>
For the treatment of the following infections caused by susceptible strains of specified microorganisms in adults (≥18 years): (1) uncomplicated urethral or cervical gonorrhea caused by Neisseria gonorrhoeae; (2) uncomplicated urinary tract infection (cystitis) caused by Escherichia coli; (3) complicated urinary tract infection caused by Escherichia coli, Staphylococcus epidermidis, or Staphylococcus saprophyticus, and complicated urinary tract infection caused by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus epidermidis, or Enterobacter cloacae.

---

Mechanism of Action>
Enoxacin exerts its bactericidal effect by inhibiting the bacterial essential enzyme DNA gyrase (DNA topoisomerase II).

---

Pharmacodynamics>
Enoxacin is a quinolone/fluoroquinolone antibiotic. Enoxacin has bactericidal activity. Its mechanism of action is to block bacterial DNA replication by binding to an enzyme called DNA gyrase, thereby preventing the DNA double helix from unwinding and thus preventing DNA replication into two double helices. Enoxacin is a broad-spectrum antibiotic that is effective against both Gram-positive and Gram-negative bacteria. Enoxacin may be effective against pathogens that develop resistance to drugs with different mechanisms of action. Enoxacin (AT-2266) is a synthetic quinolone carboxylic acid antibiotic with dual biological activities (antibacterial and anticancer/miRNA regulation) [1][5] Mechanism of action: It exerts its antibacterial effect by inhibiting bacterial DNA gyrase and topoisomerase IV, thereby blocking DNA replication/transcription. In terms of anticancer activity, it can enhance TRBP-mediated miRNA processing and RNAi efficiency, thereby inhibiting cancer cell growth[1][2][3][5]
- Antibacterial spectrum: It is effective against Gram-negative bacteria and some Gram-positive bacteria, and its efficacy is comparable to norfloxacin and superior to some β-lactam and aminoglycoside antibiotics[5]
- Therapeutic potential: It is used clinically to treat bacterial infections and has shown potential as a miRNA regulator in cancer treatment, especially suitable for TRBP-positive tumors[1][4][5]
- Target specificity: Its anticancer effect is cancer-specific and TRBP-dependent, and it has no significant inhibitory effect on normal cell proliferation[1]

C15H17FN4O3

320.32

320.128

C, 56.24; H, 5.35; F, 5.93; N, 17.49; O, 14.98

74011-58-8

84294-96-2

3229

Solid powder

1.4±0.1 g/cm3

569.9±50.0 °C at 760 mmHg

220-224ºC

298.4±30.1 °C

0.0±1.6 mmHg at 25°C

1.599

0.55

2

8

3

23

521

0

FC1C([H])=C2C(C(C(=O)O[H])=C([H])N(C([H])([H])C([H])([H])[H])C2=NC=1N1C([H])([H])C([H])([H])N([H])C([H])([H])C1([H])[H])=O

IDYZIJYBMGIQMJ-UHFFFAOYSA-N

InChI=1S/C15H17FN4O3/c1-2-19-8-10(15(22)23)12(21)9-7-11(16)14(18-13(9)19)20-5-3-17-4-6-20/h7-8,17H,2-6H2,1H3,(H,22,23)

1-ethyl-6-fluoro-4-oxo-7-piperazin-1-yl-1,8-naphthyridine-3-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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