NT-0796 - NLRP3 inhibitor

NLRP3 inflammasome inhibitor

NT-0796 is a selective inhibitor of the NLRP3 inflammasome that is more potent than MCC950 (see figure). It was shown to reverse obesity, systemic inflammation, and astrogliosis in the diet-induced obesity mouse model. Moreover, NT-0796 is showing promising results in clinical trials for its potential in treating neurodegenerative diseases (e.g. Parkinson's) and obesity-associated chronic inflammation.

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Mode of action

Inhibition of the NLRP3 inflammasome by NT-0796

NT-0796 is an ester prodrug that is converted by human carboxylesterase-1 (CES1) to the active metabolite NDT-19795, a specific inhibitor of the NLRP3 inflammasome. Due to its esterified structure, NT-0796 exhibits superior cellular uptake and enhanced tissue distribution in vivo, including passage through the blood–brain barrier. Moreover, NT-0796 exhibits greater potency than the well-known NLRP3 inhibitor MCC950 (see figure), with which it shares structural similarities (see chemical structure below); however, MCC950’s clinical development was discontinued due to unexplained liver toxicity.

Importantly, NT-0796 is only specifically active in a subset of human cell lines, like THP-1 monocytes or macrophages, which express CES1. As CES1 is absent in mice, NT-0796 requires CES1-humanized mouse models for in vivo studies. 

Key features

• Potent inhibitor of NLRP3
• Enhanced cellular uptake and bioavailability 
• Human-specific activation
• InvitroFit™ grade: each lot is highly pure (≥95%) and functionally tested

InvivoGen's NT-0796 specifically inhibits NLRP3-dependent cell death, as verified using the lactate dehydrogenase (LDH) assay LDH-Blue™ (see figures). Moreover, it successfully blocks the release of IL‑1β induced by NLRP3 activators, such as MSU or CPPD crystals, assessed using InvivoGen's THP-1 / HEK-Blue™ IL-1β release assay. Finally, NT-0796 was shown to inhibit ASC-speck formation, a marker of inflammasome assembly, using InvivoGen’s THP1-ASC-GFP cells. Finally, NT-0796 was shown to inhibit ASC-speck formation, a marker of inflammasome assembly, using InvivoGen’s THP1-ASC-GFP cells (see figures).

InvivoGen's NT-0796 is for research use only, and not for human or veterinary use.

Read our review on the Metaflammation

Figures

Inhibition of NLRP3 by NT-0796 or MCC950. THP1-Null2 cells were primed with LPS-EK (1 μg/ml) for 3 h and then stimulated with increasing concentrations of NT-0796 (pink) or MCC950 (green). After incubation with Nigericin (5 µM), cell death was assessed using LDH-Blue™. Results are presented as a percentage of the maximal LDH release measured in a positive control (mean ± SEM).

Specific inhibition of NLRP3 by NT-0796. THP1-Null2 cells were primed with LPS-EK (1 μg/ml) for 3 h and then stimulated with increasing concentrations of NT-0796 for 30 min. After overnight incubation with the Val-boroPro (VbP) + JSH-23 (CARD8 agonist, each 5 µM), Lfn-Needle (50 ng/ml) + B. anthracis protective antigen (PA) (500 ng/ml) (NAIP/NLRC4 agonist), or Nigericin (NLRP3 agonist, 5 µM), the pyroptotic cell death was assessed using the LDH-Blue™ assay. Data is shown as the percentage of cell death (mean ± SEM).

NT-0796 inhibits the NLRP3 inflammasome response in a dose-dependent manner. THP1-Null2 cells were primed with LPS-EK (1 μg/ml) for 3 h and then stimulated with increasing concentrations of NT-0796 for 30 min. After overnight incubation with MSU (250 μg/ml in red) or calcium pyrophosphate dihydrate (CPPD) crystals (25 µg/ml in blue), IL-1β secretion was analyzed by adding 50 μl of supernatant from treated THP1-Null2 cells to HEK-Blue™ IL-1β cells. IL-1β-induced activation of NF-κB was assessed by measuring the levels of SEAP in the supernatant of HEK-Blue™ IL-1β cells using QUANTI-Blue™ Solution, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm. Data are shown as percentage (%) activity.

Inhibition of NLRP3-dependent ASC-GFP speck formation. THP1-ASC-GFP cells were primed with LPS-EK (1 μg/ml) for 2 h, following stimulation with increasing concentrations of NT-0796 (0.01 nM - 10 μM). ASC-speck formation was assessed after 2 h of incubation with the NLRP3 agonist Nigericin (5 μM). ASC specks were quantified as number per field using the 10X objective (mean ± SEM). The experiment was performed in the presence of the pan-caspase inhibitor Z-VAD (10 μM) to block cell death.

Specifications

CAS number: 2272917-13-0

Working concentration: 300 nM - 10 µM

Solubility: DMSO (10 mg/ml)

Formula: C₂₃H₂₇N₃O₄

Molecular weight: 409.48 g/mol

Quality control:

• Purity: ≥95%
• The inhibitory activity of the product has been validated in inflammasome cellular assays using THP1-Null2 and HEK-Blue™ IL-1β cells.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) is confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.

Contents

NT-0796 is provided as a dried powder. It is available in two quantities:

• inh-nt0796: 1 mg
• inh-nt0796-5: 5 x 1 mg

NT-0796 is shipped at room temperature.

Upon receipt, store at -20°C for up to 2 years.

 Resuspended product is stable for up to 6 months at -20 °C when properly stored.

 Avoid repeated freeze-thaw cycles.

Details

The NLRP3 Inflammasome

The NLRP3 inflammasome is an innate immune sensor that is activated by a two-step process; a first signal (‘priming’) provided mainly by bacterial components or endogenous cytokines involves NF-κB induction, while the second signal provided by a wide array of stimuli including microbial toxins, endogenous molecules or crystalline substances and leads to inflammasome assembly and activation [1, 2].  This triggers inflammasome multimerization and caspase-1 activation with the subsequent cleavage of interleukin-1β (IL-1β)/IL-18 and the pore-forming protein Gasdermin D (GSDMD) into their active forms. Additionally, the activation of the inflammasome also leads to alarmin secretion and pyroptosis, a form of immunogenic cell death.

NT-0796 background

The NT-0796 is an ester pro-drug of the carboxylic acid active species NDT-19795 [1]. NT-0796 gets metabolized by carboxylesterase-1 (CES1) in the lumen of the endoplasmic reticulum (ER) to yield the active metabolite NDT-19795, which finally binds and inhibits the NLRP3 (NOD-like receptor pyrin domain-containing protein 3, cryopyrin, or NALP3) inflammasome [6]. Recent studies revealed that NTD-19795 binds to the NACHT domain of NLRP3, preventing the conformational changes required for inflammasome activation [3-5].

As mentioned above, CES1 is required to convert NT-0796 to the active pharmacophore NDT-19795 [3,5,6]. CES1 is abundantly expressed in the liver, as well as in human monocytes and macrophages (e.g. THP-1 cells). However, it is not found in mouse macrophages or commonly used human cell lines, such as HEK 293 cells. This makes the study design more complex and requires humanized mouse models that express human CES1 (hCES1) [5-6]. However, carboxylate esterification has many advantages since it improves bioavailability, facilitates passage across the blood-brain barrier, and enhances tissue distribution of small-molecule therapeutics [6]. NT-0796 was shown to be >100-fold more potent than NDT-19795 at inhibiting the same cellular responses. This difference in potency is attributed to neutral NT-0796 being taken up by the cells more readily than negatively charged NDT-19795 [6].

NT-0796 has proved to be a compelling, orally administrable therapeutic NLRP3 inhibitor with demonstrated in vivo CNS brain penetration [3].  It is showing promising results in clinical trials for its potential in treating neurodegenerative diseases (e.g. Parkinson's) and obesity-associated chronic inflammation [4]. Indeed, recent preclinical studies highlight NT-0796’s therapeutic potential in metabolic disease, showing that it enhances semaglutide (Wegovy)-induced weight loss while reversing hypothalamic inflammation. Moreover, combinatory treatment sustains this effect after treatment cessation, positioning it as a unique oral agent for durable weight loss, either alone or in combination with GLP-1Ras [7].

References:

1. Swanson K.V. et al., 2019. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat. Rev. Immunol. 19:477.
2. Groslambert M. & Py B. 2018. Spotlight on the NLRP3 inflammasome pathway. J. Inflamm. Res. 11:359.
3. Harrison D, et al., 2023. Discovery of Clinical Candidate NT-0796, a Brain-Penetrant and Highly Potent NLRP3 Inflammasome Inhibitor for Neuroinflammatory Disorders. J Med Chem. 9;66(21):14897-14911.
4. Thornton P, et al., 2024. Reversal of High Fat Diet-Induced Obesity, Systemic Inflammation, and Astrogliosis by the NLRP3 Inflammasome Inhibitors NT-0249 and NT-0796. J Pharmacol Exp Ther.;388(3):813-826
5. Doedens JR, et al. 2024. The ester-containing prodrug NT-0796 enhances delivery of the NLRP3 inflammasome inhibitor NDT-19795 to monocytic cells expressing carboxylesterase-1. Biochem Pharmacol. ;227:116455.
6. Smolak P, et al., 2024. Target cell activation of a structurally novel NLRP3 inhibitor NT-0796 enhances potency. Journal of Pharmacology and Experimental Therapeutics, JPET-AR-2023-001941
7. Thornton P, et al., 2025. The NLRP3 inhibitor NT-0796 enhances and sustains GLP-1R agonist-mediated weight loss in a murine diet-induced obesity model. Obesity (Silver Spring). 

Chemical structure of NT-0796