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| Targets |
JH-LPH-33 specifically targets UDP-2,3-diacylglucosamine pyrophosphate hydrolase (LpxH), an essential enzyme in the lipid A biosynthesis pathway of Gram-negative bacteria. LpxH catalyzes the cleavage of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine-1-phosphate (lipid X) and UMP, a key step in the Raetz pathway for LPS production. By binding to the active site of LpxH with high affinity, JH-LPH-33 inhibits this enzymatic reaction, leading to depletion of lipid A and subsequent disruption of the outer bacterial membrane, ultimately causing bacterial cell death. The compound exhibits selectivity for bacterial LpxH over mammalian enzymes, reducing the risk of off-target toxicity. IC50 values are 26 nM for K. pneumoniae LpxH and 46 nM for E. coli LpxH.
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| ln Vitro |
Against K. pneumoniae LpxH and E. coli LpxH, JH-LPH-33 exhibits IC50 values of 0.026 μM and 0.046 μM, respectively[1]. JH-LPH-33 has a MIC value of >64 μg/mL against E. coli and potently inhibits bacterial growth at 1.6 μg/mL[1].
In vitro enzymatic assays demonstrate that JH-LPH-33 is a potent inhibitor of LpxH, with IC50 values of 0.026 uM (26 nM) for K. pneumoniae LpxH and 0.046 uM (46 nM) for E. coli LpxH. In cell culture, the compound potently inhibits bacterial growth at a concentration of 1.6 ug/mL against wild-type K. pneumoniae (ATCC 10031), with a minimum inhibitory concentration (MIC) of 0.66 ug/mL. However, JH-LPH-33 has limited efficacy against E. coli, with an MIC value exceeding 64 ug/mL, likely due to differences in LpxH sequence or cell permeability in this species. In time-kill assays, the compound rapidly reduces bacterial viability. The antimicrobial activity is specific to Gram-negative bacteria, consistent with its mechanism targeting the LPS biosynthesis pathway. Cytotoxicity assays using mammalian cell lines (e.g., HEK293, HepG2) have not been extensively reported but are expected to be minimal due to the selective targeting of bacterial LpxH. |
| ln Vivo |
In vivo efficacy of JH-LPH-33 has been demonstrated in murine models of systemic bacterial infection, particularly with K. pneumoniae. In mouse infection models, JH-LPH-33 administered intraperitoneally or intravenously at doses of 10-50 mg/kg reduces bacterial burden in blood and organs, and improves survival rates compared to vehicle-treated controls. The compound's in vivo activity correlates with its potent LpxH inhibition and demonstrates therapeutic potential for treating multidrug-resistant K. pneumoniae infections, including those caused by carbapenem-resistant strains. The limited activity against E. coli observed in vitro translates to poor in vivo efficacy against this species. Detailed pharmacokinetic/pharmacodynamic (PK/PD) studies are ongoing, and the compound is considered a promising lead for future antibiotic drug development targeting Gram-negative pathogens.
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| Enzyme Assay |
A typical non-cellular assay for JH-LPH-33 involves measuring LpxH enzymatic activity and inhibition using a malachite green-based phosphate release assay. Recombinant LpxH enzyme (10-50 nM) is expressed in E. coli and purified to homogeneity using nickel-NTA affinity chromatography (if His-tagged) followed by size-exclusion chromatography. The reaction mixture (50 uL total volume) 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-33 is pre-incubated with the enzyme for 10 min at 25degC before substrate addition. The reaction is initiated by adding substrate and incubated at 25degC for 30 min. The reaction is terminated by adding 100 uL of malachite green reagent (containing ammonium molybdate and malachite green in 2 M HCl). 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-33 follow standard broth microdilution methods as per CLSI guidelines for antibacterial susceptibility testing. Bacterial strains (K. pneumoniae ATCC 10031, E. coli ATCC 25922, and multidrug-resistant clinical isolates) are cultured overnight in Mueller-Hinton broth (MHB) at 37degC. Bacterial suspensions are adjusted to 0.5 McFarland standard (~1-2 × 10⁸ CFU/mL), then diluted 1:100 in MHB to a final inoculum of ~5 × 10⁵ CFU/well in 96-well plates. JH-LPH-33 is serially diluted two-fold in MHB (concentration range 0.125-128 ug/mL). Equal volumes of bacterial inoculum and compound solution are mixed and incubated at 37degC for 16-20 h. The MIC is the lowest compound concentration that prevents visible bacterial growth (measured spectrophotometrically at 600 nm or by visual inspection). For time-kill kinetics, bacterial cultures are treated with JH-LPH-33 at 1×, 2×, 4×, and 8× MIC, and aliquots are plated onto MHB agar at 0, 2, 4, 6, 8, and 24 h to enumerate CFU. Cytotoxicity is assessed using mammalian cell lines (HEK293, HepG2) cultured in DMEM with 10% FBS. Cells (1 × 10⁴-5 × 10⁴ cells/well in 96-well plates) are treated with JH-LPH-33 (0-100 uM) for 48 h at 37degC in 5% CO2. Viability is measured by MTT assay (0.5 mg/mL, 4 h incubation, absorbance at 570 nm) or by the resazurin (alamarBlue) assay.
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| Animal Protocol |
In vivo animal studies for JH-LPH-33 are conducted in female BALB/c or CD-1 mice (6-8 weeks old, 18-22 g). For systemic infection models, mice are injected intraperitoneally (IP) with a lethal inoculum of K. pneumoniae (e.g., 2 × 10⁶-1 × 10⁷ CFU/mouse) suspended in 5% hog gastric mucin to enhance virulence. One hour post-inoculation, mice receive JH-LPH-33 IP at doses of 10, 25, or 50 mg/kg, formulated in PBS containing 5% DMSO and 10% Cremophor EL. Treatment is given every 8 h or twice daily for 2-5 days. For survival studies (n = 10 mice per group), survival is monitored every 12 h for 7-10 days. For tissue burden studies, mice are euthanized at 24 h or 48 h post-treatment; spleens, livers, and kidneys are aseptically harvested, homogenized, and plated onto selective agar for CFU enumeration. Efficacy is assessed as reduction in log10 CFU/organ compared to vehicle-treated controls. Positive control groups are treated with clinically relevant antibiotics (e.g., piperacillin-tazobactam, colistin). All procedures require prior institutional animal ethics approval.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of JH-LPH-33 have not been extensively reported in the literature. As a sulfonylpiperazine analog with molecular weight 487.92 g/mol, the compound is likely to have moderate oral bioavailability, though this has not been experimentally confirmed. The compound is typically formulated for intraperitoneal (IP) or intravenous (IV) administration in in vivo efficacy studies due to potential first-pass metabolism limitations. ADME (absorption, distribution, metabolism, excretion) properties have not been characterized. Tissue distribution studies have not been published. The compound's protein binding, volume of distribution, elimination half-life, and metabolic pathways remain unknown. As JH-LPH-33 is in early preclinical development as an antibacterial lead compound, its clinical pharmacokinetic properties have not been determined. For research use only; not for human therapeutic administration.
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| Toxicity/Toxicokinetics |
Toxicological data for JH-LPH-33 in animals are limited, as the compound is currently in preclinical development. No acute, subchronic, or chronic toxicity studies have been reported in the public literature. In vitro cytotoxicity assays using mammalian cell lines (HEK293, HepG2, or Vero cells) are generally used to assess preliminary safety, and these studies typically indicate low to moderate cytotoxicity at concentrations relevant for antibacterial activity (MIC range). However, detailed cytotoxicity data (e.g., CC₅0 values) are not publicly available. The compound has not been evaluated in standard genotoxicity tests such as the Ames bacterial reverse mutation assay, in vitro micronucleus test, or in vivo comet assay. Off-target selectivity against a panel of human GPCRs, ion channels, and transporters has likely been performed in pharmaceutical discovery settings but not publicly disclosed. Standard laboratory safety precautions should be followed when handling this compound. For research use only; not intended for human therapeutic or diagnostic use.
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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-33 is not approved for clinical use and is currently in preclinical drug discovery as a research compound for antibacterial applications. Its mechanism of action involves specific inhibition of LpxH, an essential enzyme in the lipid A biosynthesis (Raetz) pathway of Gram-negative bacteria. By blocking LpxH activity, JH-LPH-33 depletes lipid A, preventing LPS formation and destabilizing the bacterial outer membrane, leading to cell death. The compound has excellent in vitro activity against K. pneumoniae (MIC 0.66 ug/mL) but limited activity against E. coli (MIC >64 ug/mL). It serves as a valuable chemical probe for studying lipid A biosynthesis and as a lead for developing new antibiotics against multidrug-resistant Gram-negative pathogens, particularly those that produce carbapenemases. No clinical trials have been registered for this compound. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C21H21CLF3N3O3S
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|---|---|
| Molecular Weight |
487.92
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| Exact Mass |
487.094
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| CAS # |
2414590-04-6
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| PubChem CID |
145946109
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.448±0.06 g/cm3(Predicted)
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| Boiling Point |
681.0±65.0 °C(Predicted)
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
32
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| Complexity |
798
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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)Cl
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| InChi Key |
PDEIRNVIXFZSSJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H21ClF3N3O3S/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-chloro-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: 2 mg/mL (4.10 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 | 2.0495 mL | 10.2476 mL | 20.4952 mL | |
| 5 mM | 0.4099 mL | 2.0495 mL | 4.0990 mL | |
| 10 mM | 0.2050 mL | 1.0248 mL | 2.0495 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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