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| Targets |
DATPT targets the protein-protein interaction (PPI) between sorting nexin 9 (SNX9) and p47phox (also known as NCF1, neutrophil cytosolic factor 1). SNX9 is a member of the sorting nexin family involved in endosomal trafficking. p47phox is a regulatory subunit of the NADPH oxidase (NOX2) complex, which is responsible for superoxide production in phagocytes. By blocking the SNX9-p47phox interaction in the endosome, DATPT prevents the assembly and activation of the NOX2 complex, leading to reduced reactive oxygen species (ROS) production. Additionally, DATPT inhibits the downstream production of inflammatory cytokines, such as TNF-alpha, IL-6, and IL-1beta. The compound may also target GTP cyclohydrolase I (GTPCH1) to reduce BH4 (tetrahydrobiopterin) levels, impacting nitric oxide production and monoamine metabolism.
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| ln Vitro |
DATPT inhibits the SNX9-p47phox interaction in vitro, as demonstrated by co-immunoprecipitation (Co-IP) and pull-down assays in cell lysates. By blocking this endosomal PPI, DATPT suppresses the production of reactive oxygen species (ROS). It also suppresses the production of pro-inflammatory cytokines, including TNF-alpha, IL-6, and IL-1beta, in lipopolysaccharide (LPS)-stimulated macrophages. DATPT decreases ATP production, likely as a consequence of reduced ROS-mediated mitochondrial damage or through direct effects on energy metabolism. DATPT has antibacterial activity (unspecified mechanism), in addition to its anti-inflammatory effects. The peptide-mimetic shows low cytotoxicity in mammalian cell lines at anti-inflammatory concentrations.
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| ln Vivo |
In vivo, DATPT has demonstrated significant therapeutic effects in a mouse model of cecal ligation and puncture (CLP)-induced sepsis, a clinically relevant model of polymicrobial sepsis and septic shock. When administered intraperitoneally (i.p.) 1 hour after CLP, DATPT (doses of 10-50 mg/kg) significantly reduced serum levels of pro-inflammatory cytokines (TNF-alpha, IL-6, IL-1beta), decreased systemic oxidative stress (malondialdehyde, MDA), and improved survival rates (from ~20% in vehicle controls to ~70-80% in treated mice). DATPT also attenuated acute lung injury (ALI) and acute kidney injury (AKI) in septic mice, as assessed by histology (H&E staining) and markers of organ function (BUN, creatinine, ALT, AST). These results indicate that DATPT is a promising lead compound for sepsis therapy.
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| Enzyme Assay |
The protein-protein interaction between SNX9 and p47phox can be evaluated in cell-free systems using recombinant proteins. Recombinant GST-tagged SNX9 and His-tagged p47phox (or p47phox-derived peptide) are expressed in E. coli and purified by affinity chromatography. The binding assay is performed using glutathione-Sepharose beads. GST-SNX9 is immobilized on glutathione beads in binding buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, protease inhibitors). His-p47phox (1-5 uM) is incubated with immobilized GST-SNX9 (0.5 uM) in the presence of increasing concentrations of DATPT (0.1-100 uM) for 2 h at 4degC. Beads are washed, bound proteins are eluted with SDS sample buffer, separated by SDS-PAGE, and detected by Western blotting with anti-His antibody. The IC50 for inhibition of the SNX9-p47phox interaction is determined by densitometry. Alternatively, an ELISA-based protein-protein interaction assay can be developed: plate wells coated with recombinant SNX9 are blocked, and biotinylated p47phox is added with or without DATPT. Bound p47phox is detected by streptavidin-HRP and a colorimetric substrate. The IC50 for disruption of the PPI is calculated. Surface plasmon resonance (SPR) can also be used to measure the binding of DATPT to SNX9 or p47phox individually, but the primary mechanism is disruption of the PPI.
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| Cell Assay |
Cellular assays to evaluate DATPT are performed in mouse macrophage cell lines (e.g., RAW 264.7) or in primary peritoneal macrophages. Cells are cultured in DMEM with 10% FBS at 37degC and 5% CO2. DATPT is dissolved in DMSO and diluted in culture medium (final DMSO ≤0.1%). For ROS measurements, cells are seeded in 96-well black-walled plates (1 × 10⁵ cells/well). Cells are pre-treated with DATPT (0, 1, 10, 50 uM) for 1 h, then stimulated with lipopolysaccharide (LPS, 100 ng/mL) or phorbol myristate acetate (PMA, 100 ng/mL) for 30 min. The fluorescent probe DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate, 10 uM) is added, and fluorescence (ex/em 485/535 nm) is measured every 10 min for 60-90 min using a microplate reader. The area under the curve (AUC) is calculated as a measure of total ROS production. For cytokine measurements, cells are pre-treated with DATPT for 1 h, then stimulated with LPS (100 ng/mL) for 6-24 h. Culture supernatants are collected, and TNF-alpha, IL-6, and IL-1beta levels are measured by ELISA. Cell viability is assessed by MTT assay to ensure that the observed effects are not due to cytotoxicity. The IC50 for cytokine suppression is determined. For co-immunoprecipitation (Co-IP) assays to confirm PPI disruption in cells, cells are treated with DATPT (10-50 uM) for 6 h, then lysed in co-IP buffer. SNX9 is immunoprecipitated using an anti-SNX9 antibody, and the co-precipitation of p47phox is detected by Western blotting. DATPT treatment reduces the amount of p47phox co-precipitated with SNX9.
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| Animal Protocol |
The in vivo efficacy of DATPT is evaluated in the cecal ligation and puncture (CLP) model of sepsis in male C57BL/6 mice (8-12 weeks). Mice are anesthetized with isoflurane (2-3% in O2). A small midline abdominal incision is made, the cecum is exteriorized, and 50% of the cecum is ligated with a 4-0 silk suture distal to the ileocecal valve. The ligated cecum is punctured twice with a 21-gauge needle, and a small amount of fecal material is extruded to ensure patency. The cecum is returned to the abdomen, and the incision is closed. Immediately after surgery, mice receive an i.p. injection of 1 mL warm saline for fluid resuscitation. DATPT is dissolved in sterile saline or PBS containing 5% DMSO. At 1 h post-CLP, mice receive an i.p. injection of DATPT at doses of 0, 10, 25, or 50 mg/kg (100 uL volume). A sham-operated group (no ligation, no puncture) serves as a control. For survival studies, mice are monitored every 6 h for 7 days. Survival curves are plotted using Kaplan-Meier analysis, and the log-rank test is used to compare groups. For mechanistic studies, blood is collected at 6, 12, and 24 h post-CLP; serum is separated; and TNF-alpha, IL-6, IL-1beta, and HMGB1 are measured by ELISA. At 24 h post-CLP, mice are euthanized, and lungs, kidneys, and livers are harvested. Tissues are fixed in 10% formalin, embedded in paraffin, sectioned, and stained with H&E. Pathological scoring of injury (edema, neutrophil infiltration, alveolar septal thickening for lung; tubular necrosis for kidney; necrosis and inflammatory infiltration for liver) is performed in a blinded manner. Malondialdehyde (MDA) and glutathione (GSH) are measured in tissue homogenates to assess oxidative stress. Myeloperoxidase (MPO) activity is measured to quantify neutrophil infiltration. DATPT treatment (25 mg/kg) significantly reduces mortality (70% survival vs. 20% in vehicle, p < 0.01), decreases serum cytokines, reduces tissue oxidative stress, and improves organ histology.
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| ADME/Pharmacokinetics |
The pharmacokinetics of DATPT have not been fully characterized in published literature. As a peptide-mimetic small molecule with a molecular weight of 495.06 g/mol, DATPT is likely to have a short half-life in plasma (t½ < 2 h) due to rapid renal clearance and proteolytic degradation. The compound is soluble in DMSO (up to 50 mg/mL) and can be formulated for i.p. administration. Given the efficacy observed after i.p. injection in the CLP sepsis model, the compound is assumed to be adequately absorbed from the peritoneal cavity. No data on oral bioavailability, plasma protein binding, volume of distribution, metabolism, or excretion are available. The compound likely undergoes hepatic metabolism (CYP450) and is excreted primarily in the urine and feces. Its peptide-mimetic structure may confer some stability against protease degradation compared to a native peptide, but it is not expected to be long-acting. No PK data in humans exist.
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| Toxicity/Toxicokinetics |
DATPT exhibits a favorable safety profile in animal models. In the CLP sepsis model, doses up to 50 mg/kg (i.p.) are well-tolerated, with no additional mortality or toxicity compared to vehicle-treated mice. No studies have reported on the acute toxicity (LD50), chronic toxicity, genotoxicity, or organ-specific toxicity of DATPT. The LD50 (i.p.) is likely >200 mg/kg based on the observed tolerability. DATPT has been described as a potential therapeutic compound for sepsis, and no major adverse effects have been reported in the primary literature. Because DATPT acts by reducing ROS and inflammatory cytokines, it may have a protective effect on tissues rather than causing toxicity. Standard laboratory safety precautions (gloves, lab coat, safety glasses) should be used when handling the powder. Avoid inhalation, skin contact, and ingestion. DATPT is for research use only; it is not approved for human or veterinary therapeutic use.
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| References |
[1]. Lee D, et al. Discovery of Mycobacterium tuberculosis Rv3364c-Derived Small Molecules as Potential Therapeutic Agents to Target SNX9 for Sepsis. J Med Chem. 2022;65(1):386-408.
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| Additional Infomation |
SNX9 (sorting nexin 9) is an endosomal protein involved in membrane trafficking and actin polymerization. p47phox (NCF1, neutrophil cytosolic factor 1) is a key regulatory subunit of the NADPH oxidase (NOX2) complex that generates superoxide anions in phagocytes during the respiratory burst, important for antimicrobial defense but also causing tissue damage in sepsis and inflammatory diseases. The interaction between SNX9 and p47phox occurs in endosomes and is required for the activation of NOX2. Blocking this PPI with DATPT prevents NOX2 activation, thereby reducing ROS production and dampening the excessive inflammatory response that contributes to septic shock and organ failure. DATPT was discovered through a screening approach using a Mycobacterium tuberculosis-derived peptide (Rv3364c) as a starting point, followed by optimization to a peptide-mimetic small molecule. The compound's ability to suppress both ROS and inflammatory cytokines makes it a potential dual-action therapeutic. Sepsis is a life-threatening condition caused by a dysregulated host response to infection, leading to organ dysfunction. There are no specific small molecule therapies for sepsis; treatment is primarily supportive (fluids, vasopressors, antibiotics). DATPT represents a novel mechanism for sepsis intervention by targeting the SNX9-p47phox interaction. The compound is also being investigated for its effects on BH4 (tetrahydrobiopterin) metabolism via inhibition of GTPCH1 (GTP cyclohydrolase I). BH4 is a cofactor for nitric oxide synthase and aromatic amino acid hydroxylases. By reducing BH4 levels, DATPT may also modulate nitric oxide production, which is dysregulated in sepsis. DATPT is for research use only; it is not an FDA-approved drug.
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| Molecular Formula |
C24H39CLN6O3
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| Molecular Weight |
495.06
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| Appearance |
Typically exists as solid at room temperature
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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: 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) |
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
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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 | 2.0200 mL | 10.0998 mL | 20.1996 mL | |
| 5 mM | 0.4040 mL | 2.0200 mL | 4.0399 mL | |
| 10 mM | 0.2020 mL | 1.0100 mL | 2.0200 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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