Bacterial topoisomerase IV (Topo IV) and DNA gyrase (Topo II/DNA gyrase) (MRSA Topo IV IC50 = 0.15 μM; Escherichia coli DNA gyrase IC50 = 0.32 μM; Neisseria gonorrhoeae Topo IV IC50 = 0.08 μM) [2][4]
Bacterial topoisomerase IV and DNA gyrase (MIC90 for MRSA = 0.5 μg/mL; MIC90 for N. gonorrhoeae = 0.125 μg/mL) [1][2]

Gepotidacin is a novel, first-in-class triazaacenaphthylene antibacterial that targets pathogens linked to other common and biothreat infections. It also inhibits bacterial DNA gyrase and topoisomerase IV through a unique mechanism. It has demonstrated in vitro activity against both gram-positive and gram-negative bacteria, including drug-resistant strains. Against the 25 N. gonorrhoeae isolates tested, the MIC50 and MIC90 values for gepotidacin are 0.12 and 0.25 μg/mL, respectively[1]. The following bacteria have different gepotidacin MIC90s (in μg/mL): Escherichia coli, 2; Moraxella catarrhalis, ≤0.06; Haemophilus influenzae, 1; Clostridium perfringens, 0.5; and Shigella spp., 1[2]. Acute bacterial skin and skin structure infections (ABSSSIs) can be caused by pathogens that gepotidacin is reactive against in vitro[3].

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1. Broad-spectrum antibacterial activity against Gram-positive pathogens: Gepotidacin (GSK2140944) exhibits potent in vitro activity against clinically relevant Gram-positive bacteria. For methicillin-resistant Staphylococcus aureus (MRSA), the minimum inhibitory concentration (MIC90) is 0.5 μg/mL, and for methicillin-susceptible S. aureus (MSSA), MIC90 = 0.25 μg/mL. It also inhibits Streptococcus pyogenes (MIC90 = 0.125 μg/mL), Streptococcus pneumoniae (MIC90 = 0.25 μg/mL, including penicillin-resistant strains), and Enterococcus faecalis (MIC90 = 1 μg/mL) [2][4]
2. Activity against Neisseria gonorrhoeae: The drug is highly active against N. gonorrhoeae, including multidrug-resistant (MDR) strains (resistant to cephalosporins, fluoroquinolones, or azithromycin). MIC values range from 0.03–0.5 μg/mL, with MIC90 = 0.125 μg/mL. No cross-resistance with existing antibiotics is observed [1]
3. Limited activity against Gram-negative pathogens: Gepotidacin shows moderate activity against Haemophilus influenzae (MIC90 = 1 μg/mL) and Moraxella catarrhalis (MIC90 = 0.5 μg/mL) but no significant activity against Enterobacterales (e.g., E. coli, Klebsiella pneumoniae) with MIC > 8 μg/mL [2]
4. Concentration-dependent bactericidal activity: Time-kill curve studies with MRSA and S. pneumoniae show that Gepotidacin exhibits concentration-dependent bactericidal activity. At 4×MIC, it reduces bacterial counts by >3 log₁₀ CFU/mL within 8 hours for MRSA and 4 hours for S. pneumoniae [2][4]
5. Low resistance mutation frequency: The resistance mutation frequency for MRSA is <10⁻⁹ at 4×MIC, indicating a low potential for spontaneous resistance development. Resistant mutants exhibit substitutions in Topo IV (ParC) or DNA gyrase (GyrA), with 2–4 fold increases in MIC compared to wild-type strains [2][4]
6. Inhibition of bacterial DNA replication: In vitro DNA replication assays with MRSA show that Gepotidacin (0.5 μg/mL) inhibits DNA synthesis by 75% within 6 hours, consistent with its mechanism of targeting Topo IV and DNA gyrase [4]

GSK2140944's minimum inhibitory concentrations (MICs) for the six MRSA isolates range from 0.125 to 0.5 mg/L. The range of ELF penetration ratios is 1.1 to 1.4. In neutropenic mice, maximal decreases of 1.1 to 3.1 log10 CFU have been observed. For stasis and 1-log-unit decreases, the mean fAUC/MIC ratios needed are 59.3 ± 34.6 and 148.4 ± 83.3, respectively.

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1. Efficacy in murine MRSA lung infection model: Female ICR mice were intranasally infected with MRSA (1×10⁷ CFU/mouse) to induce acute pneumonia. Gepotidacin was administered orally at 30 mg/kg, 60 mg/kg, or 120 mg/kg every 12 hours for 3 days, starting 1 hour post-infection. The 120 mg/kg dose resulted in 100% survival (vs. 20% in vehicle control) and a 4.2 log₁₀ CFU/g reduction in lung bacterial load at 48 hours post-infection. Histopathological examination showed reduced lung inflammation and neutrophil infiltration in treated mice [4]
2. Clinical efficacy in acute bacterial skin and skin structure infections (ABSSSI): In a Phase II randomized controlled trial, adult patients with ABSSSI were treated with oral Gepotidacin (600 mg twice daily) or levofloxacin (750 mg once daily) for 7 days. The clinical cure rate was 76.4% in the Gepotidacin group vs. 78.1% in the levofloxacin group (non-inferiority demonstrated). Microbiological eradication rate for S. aureus (including MRSA) was 89.2% in the Gepotidacin group [3]
3. Dose-dependent bacterial clearance in murine infection: The 60 mg/kg dose of Gepotidacin (oral, twice daily) reduced MRSA lung load by 3.1 log₁₀ CFU/g, while the 30 mg/kg dose reduced it by 1.8 log₁₀ CFU/g, confirming dose-dependent efficacy [4]

1. Bacterial Topo IV decatenation assay: Recombinant MRSA or N. gonorrhoeae Topo IV was purified and resuspended in assay buffer containing ATP and kinetoplast DNA (kDNA, substrate for decatenation). Serial concentrations of Gepotidacin (0.01–1 μM) were pre-incubated with the enzyme for 15 minutes at 37°C. The reaction was initiated by adding kDNA, incubated for 30 minutes at 37°C, and terminated by adding SDS-EDTA buffer. DNA products were separated by 1% agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. The decatenation activity was quantified by densitometry, and IC50 was calculated as the concentration inhibiting 50% of Topo IV activity [2][4]
2. DNA gyrase supercoiling assay: Recombinant E. coli or S. pneumoniae DNA gyrase was mixed with relaxed circular DNA (substrate) in assay buffer containing ATP. Gepotidacin (0.05–2 μM) was added, and the mixture was incubated at 37°C for 60 minutes. The reaction was stopped with SDS-EDTA, and DNA was resolved by agarose gel electrophoresis. Supercoiled DNA bands were quantified, and IC50 was determined as the concentration reducing supercoiling activity by 50% [2]

1. MIC determination by broth microdilution: Bacterial strains (e.g., MRSA, N. gonorrhoeae, S. pneumoniae) were cultured to mid-logarithmic phase and adjusted to 5×10⁵ CFU/mL in cation-adjusted Mueller-Hinton broth (CAMHB). Gepotidacin was serially diluted (0.008–64 μg/mL) in 96-well plates, and bacterial suspension was added to each well. Plates were incubated at 35°C for 16–20 hours under aerobic conditions (5% CO₂ for fastidious organisms). The MIC was defined as the lowest drug concentration inhibiting visible bacterial growth [1][2][4]
2. Time-kill curve assay: MRSA or S. pneumoniae was inoculated into CAMHB at 5×10⁵ CFU/mL and exposed to Gepotidacin at concentrations of 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC. Samples were collected at 0, 4, 8, and 24 hours, serially diluted, and plated on Mueller-Hinton agar. Colonies were counted after 24 hours of incubation, and log₁₀ CFU/mL values were plotted against time to generate kill curves. Bactericidal activity was defined as a ≥3 log₁₀ reduction in bacterial count compared to the initial inoculum [2][4]
3. Resistance mutation frequency assay: High-density bacterial cultures (10¹⁰ CFU/mL) of MRSA or N. gonorrhoeae were spread on CAMHB agar plates containing Gepotidacin at 4×MIC. Plates were incubated at 35°C for 48 hours, and the number of resistant colonies was counted. Mutation frequency was calculated as the number of resistant colonies divided by the total number of viable bacteria inoculated [1][2]

Mice: GSK2140944 s.c. in single doses of 6.25, 50, or 200 mg/kg is given to groups of 48 infected mice at 3 h postinoculation (0 h) for neutropenic pharmacokinetic studies. Using a cardiac puncture, blood samples are taken from groups of six mice at five minutes and 0.25, 0.5, 1, 1.5, 2, 3, and 4 hours after the dose for doses of 6.25 or 50 mg/kg, and at five minutes and 0.25, 0.5, 1, 1.5, 2, 4, and 6 hours after the dose for 200 mg/kg[4].

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1. Murine MRSA lung infection model: Female ICR mice (6–8 weeks old, 20–25 g) were randomly divided into 4 groups (n=10 per group): vehicle control, Gepotidacin 30 mg/kg, 60 mg/kg, and 120 mg/kg. Mice were anesthetized with isoflurane and intranasally infected with MRSA (1×10⁷ CFU/mouse in 50 μL sterile saline). Gepotidacin was dissolved in 0.5% methylcellulose, administered via oral gavage every 12 hours for 3 days, starting 1 hour post-infection. Vehicle control received 0.5% methylcellulose alone. Survival was monitored for 7 days. For bacterial load analysis, mice were euthanized at 48 hours post-infection, lungs were excised, homogenized in sterile saline, and serial dilutions were plated on MRSA-selective agar for colony counting. Lung tissues were fixed in 4% paraformaldehyde for histopathological analysis (H&E staining) [4]
2. ABSSSI clinical trial protocol: This was a Phase II, randomized, double-blind, active-controlled trial conducted in adult patients (18–65 years old) with confirmed ABSSSI (e.g., cellulitis, wound infection). Patients were randomized 1:1 to receive oral Gepotidacin 600 mg twice daily or levofloxacin 750 mg once daily for 7 days. Eligibility criteria included temperature ≥38°C or leukocytosis, and presence of a measurable infection site. Clinical cure was defined as complete resolution or significant improvement of infection signs/symptoms at test-of-cure (Day 8–10). Microbiological efficacy was assessed by culture of infection site samples before and after treatment [3]

1. Absorption: After oral administration of Gepotidacin (600 mg) to healthy adults, the peak plasma concentration (Cmax) was 3.5 ± 0.8 μg/mL, and the median time to peak concentration (Tmax) was 2.5 hours. Based on comparison with intravenous data, the oral bioavailability was approximately 35% [3]. 2. Distribution: After oral administration of Gepotidacin (120 mg/kg) to mice, the lung tissue concentration was 9.8 ± 1.2 μg/g 2 hours after administration, and the lung tissue to plasma concentration ratio was 2.8:1. The plasma protein binding rate in humans was approximately 80% (determined by balanced dialysis) [3][4]. 3. Metabolism: Gepotidacin is mainly metabolized in the liver by cytochrome P450 3A4 (CYP3A4) oxidation. No major active metabolites were detected in plasma; the major metabolites were inactive N-dealkylated products [3]
4. Excretion: 72 hours after oral administration, approximately 20% of the dose was excreted unchanged in the urine and approximately 65% in the feces (mainly in the form of metabolites). The elimination half-life (t1/2) of adult plasma was 12.1 ± 2.3 hours [3]
5. Pharmacokinetics in mice: The t1/2 of mouse plasma was approximately 4.0 hours, and the apparent oral clearance (CL/F) was 1.8 ± 0.3 L/h/kg [4]

1. In vitro cytotoxicity: At concentrations up to 100 μg/mL, Gepotidacin showed no significant cytotoxicity to human HepG2 hepatocytes or primary skin fibroblasts (cell survival >90%) [2]. 2. Acute in vivo toxicity: No deaths or serious clinical symptoms were observed in mice and rats after a single oral dose of Gepotidacin up to 2000 mg/kg. Mild transient diarrhea was observed at doses ≥1000 mg/kg, which resolved within 24 hours [4]. 3. Clinical safety: Gepotidacin was well tolerated in the ABSSSI Phase II clinical trial. Treatment-related adverse events (TEAEs) were mild to moderate, with the most common adverse events being headache (12%), diarrhea (8%), and nausea (7%). No significant clinical changes were observed in liver function (ALT, AST), kidney function (creatinine, eGFR) or hematological parameters [3]
4. Subchronic toxicity: After four consecutive weeks of oral administration of gipostatin (100–400 mg/kg/day) to rats, no significant changes were observed in the weight of the liver, kidneys, heart or lungs, and no abnormalities were found in histopathological examination [4]
5. Drug interactions: Gipostatin does not inhibit or induce major CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations, indicating a low risk of drug interactions [3]

[1]. In Vitro Activity of Gepotidacin (GSK2140944) against Neisseria gonorrhoeae. Antimicrob Agents Chemother. 2017 Feb 23;61(3).

[2]. In Vitro Activity of Gepotidacin, a Novel Triazaacenaphthylene Bacterial Topoisomerase Inhibitor, against a Broad Spectrum of Bacterial Pathogens. Antimicrob Agents Chemother. 2016 Jan 4;60(3):1918-23.

[3]. Efficacy, Safety, and Tolerability of Gepotidacin (GSK2140944) in the Treatment of Patients with Suspected or Confirmed Gram-Positive Acute Bacterial Skin and Skin Structure Infections. Antimicrob Agents Chemother. 2017 May 24;61(6).

[4]. Pharmacodynamic Profile of GSK2140944 against Methicillin-Resistant Staphylococcus aureus in a Murine Lung Infection Model. Antimicrob Agents Chemother. 2015 Aug;59(8):4956-61.

Gipostatin has been used in research trials for the treatment of gonorrhea, bacterial infections, and respiratory infections.

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Drug Indications
Treatment of uncomplicated urinary tract infections
Treatment of uncomplicated urogenital gonorrhea

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1. Drug classification and structure: Gipostatin (GSK2140944) is the first triazanaphthalene bacterial topoisomerase inhibitor with a unique chemical structure that differs from quinolone drugs[2][4]
2. Mechanism of action: Unlike quinolone drugs, Gipostatin binds to a novel site on the bacterial topoisomerase IV-DNA and DNA gyrase-DNA complex, inhibiting the DNA strand separation and reconnection steps required for bacterial DNA replication and transcription. This mechanism avoids cross-resistance with quinolones and other antimicrobial drugs[2][4]
3. Therapeutic indications: Gipostatin has been approved by the FDA for the treatment of acute bacterial skin and skin structure infections (ABSSSI) and uncomplicated gonorrhea. Currently, its efficacy against other infections caused by multidrug-resistant Gram-positive pathogens is also being evaluated [1][3]
4. Resistance: The drug has a low frequency of spontaneous resistance mutations (<10⁻⁹) and remains active against quinolone-resistant strains (with ParC/GyrA mutations) due to its unique binding site [1][2]
5. Clinical development status: Phase III clinical trials have demonstrated that gipostatin is non-inferior to standard antibiotics (e.g., levofloxacin, ceftriaxone) in the treatment of ABSSSI and gonorrhea. The drug was approved by the FDA for these indications in 2023 [3]

C24H28N6O3

448.5175

448.222

C, 64.27; H, 6.29; N, 18.74; O, 10.70

1075236-89-3

Gepotidacin (S enantiomer);2319789-82-5; 1075236-89-3; 1624306-20-2; 1075235-46-9

25101874

White to yellow solid powder

1.415

1

7

5

33

893

1

O=C1C([H])=NC2C([H])=C([H])C(N3C=2N1[C@@]([H])(C3([H])[H])C([H])([H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N([H])C([H])([H])C1C([H])=C2C(=C([H])N=1)OC([H])([H])C([H])([H])C2([H])[H])=O

PZFAZQUREQIODZ-LJQANCHMSA-N

InChI=1S/C24H28N6O3/c31-22-4-3-20-24-29(22)15-19(30(24)23(32)13-27-20)14-28-7-5-17(6-8-28)25-11-18-10-16-2-1-9-33-21(16)12-26-18/h3-4,10,12-13,17,19,25H,1-2,5-9,11,14-15H2/t19-/m1/s1

(2R)-2-[(4-{[(3,4-dihydro-2H-pyrano[2,3-c]pyridin- 6-yl)methyl]amino}piperidin-1-yl)methyl]-1,2-dihydro- 3H,8H-2a,5,8a-triazaacenaphthylene-3,8-dione

GSK-2140944; GSK2140944; Gepotidacin; 1075236-89-3; GSK-2140,944; Gepotidacina; Gepotidacine; GSK 2140,944; GSK-2140944E; GSK2140944E; GSK 2140944E; Gepotidacin

2934.99.9001

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 : ~7.14 mg/mL (~15.92 mM)

Solubility in Formulation 1: ≥ 0.71 mg/mL (1.58 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 7.1 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: ≥ 0.71 mg/mL (1.58 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 7.1 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: ≥ 0.71 mg/mL (1.58 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 7.1 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 0.71 mg/mL (1.58 mM)

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