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| Targets |
JH-LPH-28 targets the bacterial enzyme UDP-2,3-diacylglucosamine pyrophosphate hydrolase (LpxH). LpxH is an essential enzyme in the biosynthesis of lipid A, a component of lipopolysaccharide (LPS) that forms the outer leaflet of the outer membrane of Gram-negative bacteria. Inhibition of LpxH prevents the formation of lipid A, thereby disrupting the integrity of the bacterial outer membrane, leading to cell death. The specificity of JH-LPH-28 for bacterial LpxH over mammalian enzymes is high, making it a promising candidate for treating Gram-negative infections, including those caused by carbapenem-resistant Enterobacteriaceae (CRE). JH-LPH-28 has IC₅0 values of 110 nM for K. pneumoniae LpxH and 83 nM for E. coli LpxH.
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| ln Vitro |
JH-LPH-28 exhibits IC50 values of 0.083 μM against E. coli LpxH and 0.11 μM against K. pneumoniae LpxH, in that order[1]. JH-LPH-28 exhibits a minimum inhibitory concentration (MIC) of 1.6 μg/mL against wild-type K. pneumoniae (ATCC 10031) and potently inhibits bacterial growth at 2.8 μg/mL[1].
In vitro, JH-LPH-28 is a highly potent LpxH inhibitor. In enzymatic assays using purified LpxH from K. pneumoniae and E. coli, the compound demonstrates IC₅0 values of 110 nM and 83 nM, respectively. The compound displays outstanding antibiotic activity in cell culture, with a minimum inhibitory concentration (MIC) of 0.83 ug/mL against wild-type K. pneumoniae. Time-kill studies show that JH-LPH-28 is rapidly bactericidal, with a ≥3 log10 reduction in bacterial counts within 2-4 hours of exposure. The compound is also active against a range of clinical isolates, including those expressing extended-spectrum beta-lactamases (ESBLs) and carbapenemases. Activity is specific to Gram-negative bacteria; it has no significant activity against Gram-positive bacteria. |
| Enzyme Assay |
A typical non-cellular LpxH assay for JH-LPH-28 is a malachite green-based phosphatase assay that measures the release of inorganic phosphate. The reaction mixture (50 uL) contains 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 0.1% Triton X-100, 10 uM UDP-2,3-diacylglucosamine (substrate), and 0.1-100 nM LpxH. JH-LPH-28 is pre-incubated with the enzyme for 10 min at 25degC before substrate addition. The reaction is initiated by adding substrate and incubated for 30 min at 25degC, then terminated with 100 uL of malachite green reagent. After 20 min of color development at room temperature, the absorbance at 620 nm is measured. The amount of released free phosphate is calculated using a phosphate standard curve. Percent inhibition is calculated relative to DMSO vehicle control. IC₅0 values are determined by fitting the inhibition data to a four-parameter logistic equation.
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| Cell Assay |
In vitro cell-based assays for JH-LPH-28 follow standard broth microdilution methods as per CLSI guidelines for antibacterial susceptibility testing. K. pneumoniae (e.g., ATCC 10031 or clinical isolates) and E. coli are cultured overnight in cation-adjusted Mueller-Hinton broth (MHB) at 37degC. The bacterial suspension is adjusted to 0.5 McFarland standard (~1-2 × 10⁸ CFU/mL) and diluted 1:100 in MHB to a final inoculum of ~5 × 10⁵ CFU/well in 96-well plates. JH-LPH-28 is serially diluted two-fold in MHB (concentration range 0.125-128 ug/mL). The plates are incubated at 37degC for 16-20 h. The MIC is defined as the lowest concentration of compound that prevents visible bacterial growth (measured spectrophotometrically at 600 nm). For time-kill kinetics, bacterial cultures are treated with JH-LPH-28 at 1×, 2×, and 4× MIC, and aliquots are plated at 0, 2, 4, 6, 8, and 24 h to enumerate CFU. Cytotoxicity is assessed using a mammalian cell line (e.g., HEK293) to determine the selectivity index.
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| Animal Protocol |
In vivo animal studies for JH-LPH-28 are conducted in female BALB/c or CD-1 mice (6-8 weeks old, 18-22 g). For a systemic infection model, mice are injected intraperitoneally (IP) with a lethal inoculum of K. pneumoniae (e.g., 2 × 10⁶ CFU/mouse) suspended in 5% hog gastric mucin. One hour post-inoculation, JH-LPH-28 is administered IP at doses of 1, 5, 10, or 20 mg/kg, formulated in PBS containing 5% DMSO and 10% Cremophor EL. Treatment is given every 8 hours for 2 days. For survival studies (n=10 mice per group), survival is monitored every 12 hours for 7 days. For tissue burden studies, mice are euthanized at 24 hours post-treatment, and spleens, livers, and kidneys are harvested, homogenized, and plated to enumerate CFU. Efficacy is assessed as reduction in log10 CFU/g compared to vehicle-treated controls.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of JH-LPH-28 are not reported in detail. Based on its chemical structure and use in mouse models, the compound is likely to have a short plasma half-life, necessitating multiple daily dosings for in vivo efficacy. In the mouse infection model, dosing every 8 hours (three times daily) was employed, suggesting rapid clearance. The compound is formulated for intraperitoneal (IP) administration, indicating potentially low oral bioavailability. Detailed PK parameters such as Cmax, Tmax, and AUC are not publicly available. As a research compound, its ADME (absorption, distribution, metabolism, excretion) properties have not been fully characterized.
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| Toxicity/Toxicokinetics |
No toxicity data is available for JH-LPH-28. In the animal efficacy studies reported, the compound was well-tolerated at the tested doses (up to 20 mg/kg IP), with no significant weight loss or gross behavioral changes observed. However, these studies were short-term (2-3 days) and not designed for a comprehensive toxicity assessment. Standard safety precautions should be used when handling this compound (gloves, lab coat, eye protection). As a bacterial LpxH inhibitor, it is not expected to have activity against mammalian enzymes, but a full toxicological evaluation would be required for any therapeutic development. For research use only; not for human therapeutic administration.
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| References |
[1]. Cho J, et al. Structural basis of the UDP-diacylglucosamine pyrophosphohydrolase LpxH inhibition by sulfonyl piperazine antibiotics. Proc Natl Acad Sci U S A. 2020 Feb 25;117(8):4109-4116.
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| Additional Infomation |
JH-LPH-28 is not approved for clinical use and is currently in preclinical development as a research antibiotic. It is a potent, targeted inhibitor of the essential Gram-negative bacterial enzyme LpxH, with an MIC of 0.83 ug/mL against K. pneumoniae. Its mechanism of action involves blocking lipid A synthesis, which disrupts the bacterial outer membrane, leading to bacterial cell death. This compound was developed as a part of a series of sulfonyl piperazine inhibitors and serves as a valuable research tool for studying LpxH biology and for validating LpxH as a therapeutic target. No clinical trials have been registered for JH-LPH-28. For research use only; not for human therapeutic or diagnostic use.
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| Molecular Formula |
C21H21F4N3O3S
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| Molecular Weight |
471.47
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| Exact Mass |
471.123
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| CAS # |
2414592-36-0
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| PubChem CID |
146014474
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.432±0.06 g/cm3(Predicted)
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| Boiling Point |
663.1±65.0 °C(Predicted)
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| LogP |
3
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
32
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| Complexity |
795
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(=O)N1CCC2=C1C=CC(=C2)S(=O)(=O)N3CCN(CC3)C4=CC(=CC(=C4)C(F)(F)F)F
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| InChi Key |
NYMCJUUEVIRRQM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H21F4N3O3S/c1-14(29)28-5-4-15-10-19(2-3-20(15)28)32(30,31)27-8-6-26(7-9-27)18-12-16(21(23,24)25)11-17(22)13-18/h2-3,10-13H,4-9H2,1H3
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| Chemical Name |
1-[5-[4-[3-fluoro-5-(trifluoromethyl)phenyl]piperazin-1-yl]sulfonyl-2,3-dihydroindol-1-yl]ethanone
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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 |
| 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: 12.5 mg/mL (26.51 mM)
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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 | 2.1210 mL | 10.6051 mL | 21.2103 mL | |
| 5 mM | 0.4242 mL | 2.1210 mL | 4.2421 mL | |
| 10 mM | 0.2121 mL | 1.0605 mL | 2.1210 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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