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| Targets |
Bacterial topoisomerase IV (Topo IV) and DNA gyrase (Topo II/DNA gyrase)
Bacterial type II topoisomerases: DNA gyrase and topoisomerase IV. Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). It selectively inhibits topoisomerase IV and the B subunit of DNA gyrase. [1, 2] Gepotidacin induces high levels of gyrase-mediated single-stranded DNA breaks, in contrast to fluoroquinolones which induce primarily double-stranded breaks. [1] |
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| ln Vitro |
Gepotidacin is a potent inhibitor of S. aureus gyrase-catalyzed DNA supercoiling with an IC₅₀ of approximately 0.047 μM, and relaxation of positively supercoiled substrates with an IC₅₀ of approximately 0.6 μM. In comparison, moxifloxacin showed IC₅₀ values of approximately 11.5 μM (supercoiling) and 73 μM (relaxation). [1]
Gepotidacin enhances gyrase-mediated single-stranded DNA cleavage with an EC₅₀ of approximately 0.13 μM (in the absence of ATP) and 0.04 μM (in the presence of 1.5 mM ATP). No double-stranded DNA breaks were observed even at high compound concentrations, extended reaction times, or in the presence of ATP. [1] Gepotidacin forms stable gyrase-DNA cleavage complexes that persist for >4 hours. [1] Gepotidacin suppresses double-stranded DNA breaks generated by S. aureus gyrase, as demonstrated by a coordinate decrease in double-stranded breaks with increasing compound concentration in the presence of Ca²⁺. [1] Gepotidacin and moxifloxacin binding in the gyrase active site are mutually exclusive; increasing concentrations of gepotidacin (0-100 μM) reduced moxifloxacin-induced double-stranded breaks by approximately 95%. [1] Gepotidacin demonstrates potent in vitro activity against a broad spectrum of bacterial pathogens. MIC₉₀ values: MRSA (0.5 mg/L), MSSA (0.5 mg/L), S. pneumoniae (0.25 mg/L), S. pyogenes (0.25 mg/L), H. influenzae (1 mg/L), M. catarrhalis (≤0.06 mg/L), E. coli (2 mg/L), Shigella spp. (0.5 mg/L), C. perfringens (0.5 mg/L), N. gonorrhoeae (0.5 mg/L). For levofloxacin-non-susceptible E. coli isolates, the MIC₉₀ increased to 4 mg/L. [2] Gepotidacin inhibits 90% of Gram-negative anaerobes at 4 mg/L and Gram-positive anaerobes at 2 mg/L. [2] In vitro studies show activity against mycoplasma and ureaplasma infections, including drug-resistant Mycoplasma genitalium. [2] Gepotidacin shows activity against mycobacterial pathogens, including Mycobacterium tuberculosis and drug-resistant non-tuberculosis mycobacteria. [2] Gepotidacin is a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC50 ≈ 0.047 μM) and relaxation of positively supercoiled substrates (IC50 ≈ 0.6 μM) [1]. The MICs of gepotidacin against E. coli isolates in Mueller-Hinton broth range from 1 to 4 mg/L [2]. |
| ln Vivo |
Gepotidacin is a potent inhibitor of S. aureus gyrase-catalyzed DNA supercoiling with an IC₅₀ of approximately 0.047 μM, and relaxation of positively supercoiled substrates with an IC₅₀ of approximately 0.6 μM. In comparison, moxifloxacin showed IC₅₀ values of approximately 11.5 μM (supercoiling) and 73 μM (relaxation). [1]
Gepotidacin enhances gyrase-mediated single-stranded DNA cleavage with an EC₅₀ of approximately 0.13 μM (in the absence of ATP) and 0.04 μM (in the presence of 1.5 mM ATP). No double-stranded DNA breaks were observed even at high compound concentrations, extended reaction times, or in the presence of ATP. [1] Gepotidacin forms stable gyrase-DNA cleavage complexes that persist for >4 hours. [1] Gepotidacin suppresses double-stranded DNA breaks generated by S. aureus gyrase, as demonstrated by a coordinate decrease in double-stranded breaks with increasing compound concentration in the presence of Ca²⁺. [1] Gepotidacin and moxifloxacin binding in the gyrase active site are mutually exclusive; increasing concentrations of gepotidacin (0-100 μM) reduced moxifloxacin-induced double-stranded breaks by approximately 95%. [1] Gepotidacin demonstrates potent in vitro activity against a broad spectrum of bacterial pathogens. MIC₉₀ values: MRSA (0.5 mg/L), MSSA (0.5 mg/L), S. pneumoniae (0.25 mg/L), S. pyogenes (0.25 mg/L), H. influenzae (1 mg/L), M. catarrhalis (≤0.06 mg/L), E. coli (2 mg/L), Shigella spp. (0.5 mg/L), C. perfringens (0.5 mg/L), N. gonorrhoeae (0.5 mg/L). For levofloxacin-non-susceptible E. coli isolates, the MIC₉₀ increased to 4 mg/L. [2] Gepotidacin inhibits 90% of Gram-negative anaerobes at 4 mg/L and Gram-positive anaerobes at 2 mg/L. [2] In vitro studies show activity against mycoplasma and ureaplasma infections, including drug-resistant Mycoplasma genitalium. [2] Gepotidacin shows activity against mycobacterial pathogens, including Mycobacterium tuberculosis and drug-resistant non-tuberculosis mycobacteria. [2] |
| Enzyme Assay |
DNA Supercoiling and Relaxation Assays: Reactions contained 20 nM S. aureus gyrase, 5 nM relaxed or positively supercoiled pBR322 DNA, 1.5 mM ATP, 1 mM dithiothreitol in buffer (50 mM Tris-HCl pH 7.7, 20 mM KCl, 300 mM KGlu, 5 mM MgCl₂, 0.05 mg/mL BSA). Reactions were incubated at 37°C for 25 min (supercoiling) or 0.5 min (relaxation). Reactions were stopped with SDS/EDTA, treated with proteinase K, and analyzed by agarose gel electrophoresis. IC₅₀ values were calculated using nonlinear regression. [1]
DNA Cleavage Assays: Reactions contained 75 nM S. aureus gyrase (A₂B₂) and 10 nM supercoiled pBR322 in cleavage buffer (50 mM Tris-HCl pH 7.5, 100 mM KGlu, 5 mM MgCl₂ or CaCl₂, 1 mM DTT, 50 μg/mL BSA). Reactions were incubated at 37°C for 30 min. Enzyme-DNA cleavage complexes were trapped by adding SDS, EDTA, and proteinase K, followed by incubation at 45°C for 30 min. Products were analyzed by agarose gel electrophoresis containing 0.5 μg/mL ethidium bromide. EC₅₀ values were calculated using nonlinear regression. [1] Cleavage Complex Stability Assay: Initial reactions (375 nM gyrase, 50 nM DNA, 5 μM gepotidacin or 25 μM moxifloxacin) were incubated at 37°C for 30 min, then diluted 20-fold with buffer lacking divalent metal ion. Samples were removed at times from 0-240 min, and DNA cleavage was stopped and processed as above. [1] |
| Animal Protocol |
Rat Pyelonephritis Model: A rat model of MDR E. coli pyelonephritis was used with recreated human drug exposure of gepotidacin to evaluate efficacy. [2]
Non-Human Primate Plague Model: A primate animal model was used to demonstrate efficacy of gepotidacin against Yersinia pestis. [2] |
| ADME/Pharmacokinetics |
Following a single oral dose of gepotidacin (1500 mg mesylate salt capsule) in healthy fasting adults, mean AUC₀-∞ was 15.8 μg•h/mL, mean Cmax was 4.37 μg/mL, and mean terminal elimination t₁/₂ was 11.8 h. Similar values were observed with free-base tablet formulations. [2]
Plasma concentrations peak at 3.00 h with a single dose. Two plasma concentration peaks were observed at 1.5 h and 2.25 h after two doses administered 12 or 6 h apart. [2] Approximately 50% of an oral dose is absorbed, and about 20% of the dose is eliminated unchanged in the urine. Gepotidacin urine concentrations >4 mg/L were maintained for 24 h after a 1500 mg dose. [2] The PK/PD index associated with efficacy is similar to that of fluoroquinolones. The median gepotidacin free-drug AUC/MIC ratios associated with net bacterial stasis and 1- and 2-log₁₀ CFU reductions were 33.9, 43.7, and 60.7, respectively. Free-drug AUC/MIC ratios of ≥275 were sufficient to suppress microbial resistance. [2] |
| Toxicity/Toxicokinetics |
In healthy volunteers, IV doses of 1000 and 1800 mg gepotidacin caused a mild increase in resting heart rate (7-10 beats/min) and slight QT prolongation. [2]
In Phase II trials, the most common adverse events were gastrointestinal: nausea (20%), diarrhea (13%) in ABSSSI trials; diarrhea (27%), flatulence (23%), abdominal pain (15%), nausea (13%) in gonorrhea trials. In a UTI Phase II trial, 21/22 participants had gastrointestinal adverse events, mainly diarrhea (82%), nausea (77%), and vomiting (23%). [2] Gastrointestinal side effects are dose-related in prevalence and severity. Emesis shortly after administration did not significantly impact plasma concentrations. [2] Gepotidacin was safe and generally well tolerated in subjects with normal or impaired hepatic function. Dosing adjustments are not likely necessary for mild to moderate hepatic impairment; severe impairment may require increased dosing interval or dose reduction. [2] No clinically significant ECG findings or changes from baseline (QTcF ≥480 ms or increase >30 ms) were observed in a Phase II UTI trial. [2] |
| References |
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| Additional Infomation |
Gepotidacin (previously GSK2140944) is a first-in-class, bactericidal, oral triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by blocking two essential topoisomerase enzymes (DNA gyrase and topoisomerase IV). Mutations in both enzymes would likely be necessary for resistance to occur. [2]
Gepotidacin has successfully completed Phase II trials for acute bacterial skin/skin structure infections, uncomplicated urogenital gonorrhea, and uncomplicated urinary tract infections. Phase III trials (EAGLE-1, EAGLE-2, EAGLE-3) are underway or completed. The EAGLE-2 and EAGLE-3 trials comparing gepotidacin with nitrofurantoin for uncomplicated UTI were halted early due to strong efficacy data. [2] If approved, gepotidacin will be the first new oral antibiotic for UTIs in more than 20 years. [2] Two crystal structures of gepotidacin with S. aureus gyrase core fusion truncate were determined: one with nicked DNA (2.31 Å resolution) and one with intact DNA (2.37 Å resolution). A single gepotidacin molecule binds midway between the two scissile DNA bonds (pocket 2D) and in a pocket between the two GyrA subunits (pocket 2A). The central linker of gepotidacin shows conformational flexibility, which may contribute to its activity. [1] |
| Molecular Formula |
C25H36N6O8S
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|---|---|
| Molecular Weight |
580.65
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| Exact Mass |
580.2315
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| Elemental Analysis |
C, 51.71; H, 6.25; N, 14.47; O, 22.04; S, 5.52
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| CAS # |
1624306-20-2
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| Related CAS # |
1075236-89-3; 2319789-82-5; 1624306-20-2; 1075235-46-9
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| PubChem CID |
91668189
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| Appearance |
Off-white to light yellow solid powder
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
40
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| Complexity |
985
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1CC2=CC(CNC3CCN(C[C@@H]4CN5C(=O)C=CC6N=CC(=O)N4C5=6)CC3)=NC=C2OC1.S(C)(O)(=O)=O.O.O
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| InChi Key |
MTLHHQWYERWLIX-RGFWRHHQSA-N
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| InChi Code |
InChI=1S/C24H28N6O3.CH4O3S.2H2O/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;1-5(2,3)4;;/h3-4,10,12-13,17,19,25H,1-2,5-9,11,14-15H2;1H3,(H,2,3,4);2*1H2/t19-;;;/m1.../s1
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| Chemical Name |
(3R)-3-[[4-(3,4-dihydro-2H-pyrano[2,3-c]pyridin-6-ylmethylamino)piperidin-1-yl]methyl]-1,4,7-triazatricyclo[6.3.1.04,12]dodeca-6,8(12),9-triene-5,11-dione;methanesulfonic acid;dihydrate
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| Synonyms |
Gepotidacin mesylate; Gepotidacin mesylate dihydrate; Gepotidacin mesylate [USAN]; UNII-5P7X0H2O6B; 1624306-20-2;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~250 mg/mL (~430.55 mM; with ultrasonication)
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| Solubility (In Vivo) |
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 Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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)] Oral Formulations
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 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7222 mL | 8.6110 mL | 17.2221 mL | |
| 5 mM | 0.3444 mL | 1.7222 mL | 3.4444 mL | |
| 10 mM | 0.1722 mL | 0.8611 mL | 1.7222 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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