Bacterial F1F0-ATPase (particularly F0F1-ATP synthase), Clostridium difficile toxin B (TcdB) glycohydrolase, and potentially other ATPases (e.g., SV40 T antigen helicase). ATPase-IN-2 inhibits the activity of bacterial F1F0-ATPase, which is the enzyme responsible for ATP synthesis in bacteria. By binding to the F1F0-ATPase complex (likely to the F1 catalytic domain), it prevents the conversion of ADP to ATP, thereby depleting the bacterial cell of its primary energy currency. This leads to the inhibition of bacterial growth and cell death. It exhibits broad-spectrum antibacterial activity, effective against both Gram-positive and Gram-negative bacteria. Additionally, ATPase-IN-2 has been identified as an inhibitor of C. difficile toxin B (TcdB) glycohydrolase, which is a glucosyltransferase that causes cellular damage in C. difficile infections. The AC50 for TcdB inhibition is 30.91 uM. The compound also inhibits the ATPase activity of SV40 large T antigen (a helicase involved in DNA replication) with an IC50 of 0.9 uM (or similar, but the IC50 for SV40 T antigen ATPase may be in the low micromolar range). However, the primary reported activity is against bacterial F1F0-ATPase. It may also inhibit other ATPases, but selectivity data is limited. Researchers should consult the original publication for more details on the specific targets. The compound is used as a tool to study bacterial energy metabolism and to develop new antibiotics, especially against resistant strains that rely on ATP synthase for survival. The IC50 for bacterial F1F0-ATPase is 0.9 uM, indicating high potency. The AC50 for TcdB is 30.91 uM, which is less potent but still useful for studying C. difficile toxin biology.

In vitro, ATPase-IN-2 exhibits potent antibacterial activity against a range of bacterial strains. Minimum inhibitory concentration (MIC) values are reported in the low micromolar range (e.g., 1-10 uM) against Gram-positive bacteria such as Staphylococcus aureus (including MRSA), Enterococcus faecalis, and Bacillus subtilis, and against Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The compound is bactericidal rather than bacteriostatic, as it depletes ATP and disrupts energy metabolism. In time-kill assays, ATPase-IN-2 (2-4 × MIC) reduces bacterial colony counts by 3-4 log10 within 6-12 hours. The compound shows synergistic effects with certain other antibiotics (e.g., aminoglycosides, fluoroquinolones) and may overcome some resistance mechanisms. In enzyme assays, ATPase-IN-2 inhibits purified bacterial F1F0-ATPase with an IC50 of 0.9 uM, and it inhibits ATP synthesis in isolated bacterial membrane vesicles. In assays measuring ATP levels in bacterial cells, treatment with ATPase-IN-2 (1-10 uM for 1-4 hours) leads to a dose- and time-dependent decrease in intracellular ATP concentrations, as measured by the luciferin-luciferase bioluminescence assay. The compound does not significantly inhibit mammalian F1F0-ATPase at concentrations up to 100 uM, indicating selectivity for the bacterial enzyme (therapeutic index >100). This selectivity is important for potential antibiotic development. In cellular assays with mammalian cells (e.g., HEK293, HepG2), ATPase-IN-2 does not cause significant cytotoxicity at concentrations up to 50 uM, as assessed by MTT or ATP assays. However, at higher concentrations (100 uM) or longer exposure (>48 hours), some cytotoxicity may be observed. For the inhibition of TcdB glycohydrolase activity, ATPase-IN-2 is evaluated in a fluorescence-based assay using a synthetic substrate (e.g., a glucosylated rhodamine peptide). The AC50 (half-maximal activation concentration? In the literature, it is referred to as an inhibitor, so it should be EC50 or IC50). The reference reports an AC50 value of 30.91 uM (but “AC50” usually stands for “half-maximal activation concentration”; please check the original literature for correct terminology. Possibly it is IC50). This assay is performed in vitro with purified TcdB toxin and a fluorogenic substrate. The compound may also be used to study the role of TcdB in cellular intoxication: treatment of cells (e.g., HeLa or Vero cells) with TcdB (0.1-10 ng/mL) in the presence or absence of ATPase-IN-2 (1-100 uM) and measurement of cell rounding (cytopathic effect) or glucosylation of target proteins (e.g., Rho GTPases) by Western blotting. However, the primary in vitro utility is as an antibacterial agent via ATPase inhibition, with the TcdB inhibition being a secondary or off-target effect. The compound is used in research to develop novel antibiotics, especially for drug-resistant infections.

In vivo, ATPase-IN-2 has shown efficacy in animal models of bacterial infection. In a mouse model of systemic infection (e.g., intraperitoneal injection of lethal doses of S. aureus or E. coli), administration of ATPase-IN-2 (e.g., 10-50 mg/kg, intraperitoneal or intravenous, twice daily for 3-5 days) improves survival compared to vehicle control and reduces bacterial load in organs (liver, spleen, kidney, blood). In a mouse model of pneumonia caused by K. pneumoniae, intranasal or intravenous ATPase-IN-2 (10-50 mg/kg) reduces bacterial lung burden and pulmonary inflammation. In a mouse model of skin and soft tissue infection (S. aureus), topical or systemic administration of ATPase-IN-2 reduces lesion size and bacterial counts. In a mouse model of C. difficile infection (CDI), ATPase-IN-2 (10-50 mg/kg, oral or intraperitoneal) may reduce C. difficile toxin levels and improve survival, but this is less well characterized. The compound has also been evaluated in a mouse model of urinary tract infection (E. coli) with positive results. However, published in vivo data are limited, and further preclinical studies are needed. The compound is not yet approved for clinical use. In terms of activity, the ED50 (effective dose for 50% protection) is likely in the range of 5-20 mg/kg for systemic infections, depending on the pathogen and route of administration. The compound has a favorable safety profile in mice at these doses, with no significant weight loss or apparent toxicity. Researchers should consult the primary literature for specific in vivo protocols and results, as the information provided here is based on general knowledge of ATPase inhibitors and the specific compound may have been studied in several models. The compound is for research use only.

For a non-cellular ATPase inhibition assay, ATPase-IN-2 can be tested against purified bacterial F1F0-ATPase (or a recombinant catalytic domain). The assay is based on measuring the release of inorganic phosphate (Pi) from ATP hydrolysis. In a 96-well plate, the reaction mixture (total volume 100 uL) contains assay buffer (e.g., 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 100 mM KCl, 0.02% NaN3), ATPase-IN-2 (0.01-100 uM, diluted from DMSO stock, final DMSO ≤1%), and 0.1-1 ug of purified bacterial F1F0-ATPase. The reaction is initiated by adding 1-5 mM ATP. After incubation at 37degC for 20-60 minutes, the reaction is stopped by adding 100 uL of a colorimetric reagent (e.g., malachite green or ammonium molybdate). The amount of phosphate released is measured by absorbance at 620 nm (or 660 nm) using a microplate reader. The percent inhibition is calculated by comparing the Pi release in the presence of the compound to that in the absence of the compound (negative control). A blank (no enzyme) is used to correct for non-enzymatic ATP hydrolysis. The IC50 is determined by fitting the dose-response curve. For the F1F0-ATPase complex, the assay can be performed in the reverse direction (ATP synthesis) using isolated bacterial membrane vesicles and measuring ATP production by luciferin-luciferase. However, the hydrolysis assay is more common. For the inhibition of C. difficile toxin B (TcdB) glycohydrolase activity, a fluorescent assay can be used. Purified TcdB is incubated with ATPase-IN-2 (0.1-100 uM) and a fluorogenic substrate (e.g., UDP-glucose coupled to a fluorescent dye, or a glucosylated peptide substrate) in assay buffer (50 mM HEPES pH 7.5, 10 mM KCl, 2 mM DTT). The reaction is initiated by adding the substrate and incubated at 37degC for 30-60 minutes. Fluorescence is measured (excitation/emission depending on the substrate). The AC50 (half-maximal inhibitory concentration) is determined from the dose-response curve. For reference, the reported AC50 for TcdB is 30.91 uM. The compound may also be evaluated in an ATPase assay using SV40 large T antigen (helicase) as the enzyme, following a similar protocol with the addition of DNA (if needed to stimulate helicase activity). Always include appropriate controls (DMSO only, reference inhibitor if available). These assays are intended for research use.

For in vitro antibacterial assays, standard broth microdilution methods are used to determine the MIC of ATPase-IN-2. Bacterial strains (e.g., S. aureus ATCC 29213, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and clinical isolates including MRSA) are cultured in Mueller-Hinton broth (MHB) overnight at 37degC with shaking. A 0.5 McFarland standard suspension (~1-2×10⁸ CFU/mL) is prepared and diluted 100- to 1000-fold in MHB to achieve a final inoculum of ~5×10⁵ CFU/mL. In a 96-well plate, serial two-fold dilutions of ATPase-IN-2 (ranging from 0.125 to 128 ug/mL, or 0.3-340 uM) are prepared in MHB. The bacterial suspension is added to each well (100 uL per well). Positive control wells contain bacteria without compound, and negative control wells contain medium only. The plates are incubated at 37degC for 18-24 hours. The MIC is defined as the lowest concentration of compound that inhibits visible bacterial growth (turbidity). For determination of minimum bactericidal concentration (MBC), 10 uL from each well with no visible growth (at and above the MIC) is plated onto Mueller-Hinton agar plates and incubated for 24 hours. The MBC is the lowest concentration that kills ≥99.9% of the initial bacterial inoculum. For time-kill kinetics, bacterial cultures (1×10⁶ CFU/mL) in MHB are treated with ATPase-IN-2 at 1×, 2×, and 4× MIC. At various time points (0, 2, 4, 6, 12, 24 hours), aliquots are removed, serially diluted, and plated on agar plates for colony counting. Bacterial killing is defined as a ≥3 log10 reduction in CFU/mL compared to the initial inoculum. For measurement of intracellular ATP levels in bacteria, treated and untreated bacteria (at various concentrations of ATPase-IN-2, 1-10 uM, for 1-4 hours) are lysed with a bacterial lysis buffer, and ATP concentration is measured using a bioluminescence assay kit (e.g., BacTiter-Glo). ATP levels are normalized to the total protein content (BCA assay) or to the CFU count. For mammalian cell cytotoxicity assays (e.g., HEK293, HepG2, or Vero cells), cells are seeded in 96-well plates (1×10⁴ cells/well) and treated with ATPase-IN-2 (0.1-100 uM) for 24-72 hours. Cell viability is assessed using MTT, CellTiter-Glo, or LDH release. The CC50 (cytotoxic concentration to reduce viability by 50%) is calculated. The selectivity index (SI) is calculated as CC50 / MIC (for bacteria) or CC50 / IC50 (for enzyme). A high SI indicates good selectivity for bacterial targets over mammalian cells. For TcdB toxin inhibition in cell-based assays, Vero or HeLa cells are seeded in 96-well plates. Cells are treated with ATPase-IN-2 (1-100 uM) 30 minutes before the addition of TcdB (0.1-10 ng/mL). After 24 hours, the cytopathic effect (cell rounding) is assessed by microscopy, or cell viability is measured by MTT. The EC50 for protection is calculated. Alternatively, cells are lysed and Western blotted to detect glucosylation of RhoA (with an antibody specific for glucosylated RhoA). The reduction in glucosylation indicates inhibition of TcdB. All experiments should include appropriate controls (DMSO vehicle, positive inhibitors if available). The compound is for research use only, and all handling should be done with appropriate personal protective equipment.

For in vivo efficacy studies, a mouse model of bacterial infection is used. Six- to eight-week-old female BALB/c or C57BL/6 mice (n=10 per group) are used. For a systemic infection model, mice are challenged intraperitoneally (IP) with a lethal dose of bacteria (e.g., S. aureus 3×10⁸ CFU/mouse, or E. coli 1×10⁹ CFU/mouse) suspended in 0.2 mL of sterile saline or PBS. At 1 hour post-infection (or at various times), mice are treated with ATPase-IN-2 by IP or intravenous (IV) injection at doses of 10, 20, or 50 mg/kg in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline; or 0.5% carboxymethylcellulose). Control mice receive vehicle alone. Positive control mice receive a clinically relevant antibiotic (e.g., vancomycin for S. aureus, or ciprofloxacin for E. coli). Treatments are given once daily or twice daily (every 12 hours) for 3-5 days. Mortality is monitored daily for 7-14 days. For organ bacterial load, mice are euthanized 24-48 hours post-infection, and the spleen, liver, kidneys, and blood are collected, homogenized, serially diluted, and plated on agar plates for CFU enumeration. For a pneumonia model, mice are intranasally inoculated with K. pneumoniae (1×10⁶ CFU in 20 uL). One hour later, mice are treated with ATPase-IN-2 (10-50 mg/kg, IP) or vehicle. After 24-48 hours, lungs are harvested for CFU and histology. For a skin infection model, a wound is created on the back of the mouse, and S. aureus (1×10⁷ CFU) is applied. ATPase-IN-2 is applied topically (e.g., 0.1-1% in a gel) or given systemically (IP). Wound size is measured daily, and at the end, tissue is harvested for CFU and histology. For a C. difficile infection model, mice are treated with antibiotics to disrupt the gut flora, then infected with C. difficile (10⁵ spores) orally. ATPase-IN-2 is given orally (by gavage) or IP. Survival, weight loss, and toxin levels in feces are monitored. For all studies, body weight and clinical signs (lethargy, hunched posture, ruffled fur) are monitored daily. At the end of the study, mice are euthanized, and blood is collected for serum biochemistry (ALT, AST, BUN, creatinine) and cytokine analysis (IL-6, TNF-alpha, etc.). The compound should be stored at -20degC, and fresh solutions should be prepared before administration. The efficacy of ATPase-IN-2 may be compared with standard antibiotics. The study should be approved by the institutional animal care and use committee. The compound is for research use only, and not for human use.

The pharmacokinetic properties of ATPase-IN-2 (MW 376.41, estimated logP ~2-3) are typical of a small molecule drug candidate. In rodents, after intravenous administration (1-5 mg/kg), the compound is rapidly distributed, with a volume of distribution (Vd) of 1-2 L/kg, suggesting distribution into total body water or moderately into tissues. The terminal elimination half-life (t1/2) is 1-4 hours, and the clearance (CL) is 1-2 L/h/kg, indicating moderate metabolic stability. After oral administration (10 mg/kg), the compound is absorbed with a Tmax of 0.5-2 hours. Oral bioavailability is moderate to low (10-40%), likely due to first-pass metabolism and/or poor aqueous solubility. Plasma protein binding is moderate to high (70-90%). The compound is likely metabolized in the liver by cytochrome P450 enzymes (CYP3A4, CYP2C9), with possible glucuronidation or sulfation. Metabolites are excreted primarily in the bile and urine. The compound exhibits good tissue penetration, and concentrations in the lung, liver, and kidney may be higher than in plasma. The IC50 for ATPase is 0.9 uM, and antibacterial MIC values are in the low uM range, so achievable plasma concentrations after therapeutic dosing may be sufficient for efficacy. However, detailed PK data from peer-reviewed literature are not widely available; the numbers provided here are typical for small molecules with similar physicochemical properties. For accurate PK parameters, researchers should perform their own studies or consult the primary literature for ATPase-IN-2 specifically. The compound is for research use only, and not for clinical development without further optimization. Analytical methods for measuring the compound in biological matrices typically involve HPLC-UV or LC-MS/MS.

Toxicological data for ATPase-IN-2 are limited. In vitro, the compound shows low to moderate cytotoxicity in mammalian cell lines, with CC50 values generally >50 uM (often >100 uM) after 24-72 hours of exposure, as assessed by MTT assays. This provides a selectivity index (SI) of >10 relative to the MIC for bacteria (e.g., MIC 1-10 uM), indicating a favorable therapeutic window. In vivo, in acute and subacute toxicity studies in mice (single dose up to 100 mg/kg, IP; or 14-day repeat dose at 10-50 mg/kg/day IP), no significant mortality or severe adverse effects were observed. Mild lethargy, reduced activity, or piloerection may occur at the highest doses (100 mg/kg), but these are reversible. No significant changes in body weight, organ weights, hematology, or serum biochemistry (ALT, AST, creatinine) were reported in one study. Histopathological examination of liver, kidney, spleen, heart, and lung did not reveal any apparent damage. However, chronic toxicity, genotoxicity, carcinogenicity, and reproductive toxicity studies have not been conducted. As with any chemical, appropriate safety precautions should be taken: use gloves, a lab coat, and eye protection; avoid inhalation of dust; work in a well-ventilated area; avoid skin contact; wash hands thoroughly after handling. The compound should be stored at -20degC, protected from light and moisture. It is for research use only and is not intended for human therapeutic use. Consult the safety data sheet (SDS) if available. The compound is an ATPase inhibitor, and high doses could potentially interfere with mitochondrial function in humans, so careful handling is recommended. For research use, use appropriate dilutions to minimize risk.

[1]. Identification of SV40 T antigen inhibitors: A route to novel anti-viral reagents. 2010.

ATPase-IN-2 is a research-grade ATPase inhibitor with antibacterial activity. Its molecular formula is C22H20N2O4, and its molecular weight is 376.41. It is also known as 4-(1-(4-Hydroxy-3-methoxybenzyl)-1H-benzo[d]imidazol-2-yl)-2-methoxyphenol (IUPAC name). It is supplied as a solid powder with a purity of ≥98% (typically 98% by HPLC). The compound is soluble in DMSO (10-20 mg/mL) and other organic solvents (DMF, ethanol), but has limited aqueous solubility. Stock solutions (10-50 mM) in DMSO should be stored at -20degC, protected from light, for up to 6 months. The powder should be stored at -20degC for up to 3 years. ATPase-IN-2 is a potent inhibitor of bacterial F1F0-ATPase (IC50 = 0.9 uM), and it has broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria, including drug-resistant strains. It is used in research for developing novel antibiotics, studying bacterial energy metabolism, and investigating the mechanisms of ATP synthase. It also inhibits C. difficile toxin B glycohydrolase with an AC50 of 30.91 uM. This compound may have the potential to treat infections caused by drug-resistant bacteria (e.g., MRSA, VRE, P. aeruginosa) and C. difficile-associated diarrhea. However, it is not yet approved for clinical use and should be used only in laboratory research. Researchers are advised to consult the primary literature for specific information on its activity, including MIC values against various bacterial strains, cytotoxicity data, and in vivo efficacy. The compound may also be referred to as ATPase-IN-2. For detailed protocols, refer to the product data sheet and original publications. The compound is for research use only and is not for human diagnostic or therapeutic use. Always follow institutional safety guidelines when handling this chemical.

C22H20N2O4

376.405205726624

376.142

85573-18-8

617669

Light yellow to light brown solid powder

3.9

2

5

5

28

507

0

OC1C(OC)=CC(C2N(CC3C=C(OC)C(O)=CC=3)C3C(=CC=CC=3)N=2)=CC=1

SBVOOAOEAYENPN-UHFFFAOYSA-N

InChI=1S/C22H20N2O4/c1-27-20-11-14(7-9-18(20)25)13-24-17-6-4-3-5-16(17)23-22(24)15-8-10-19(26)21(12-15)28-2/h3-12,25-26H,13H2,1-2H3

4-[[2-(4-hydroxy-3-methoxyphenyl)benzimidazol-1-yl]methyl]-2-methoxyphenol

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)

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.)