Modeling-Enabled Characterization of Novel NLRX1 Ligands

Area of Science:

• Immunology and Molecular Biology
• Natural Product Chemistry
• Inflammation Research

Background:

• Nucleotide-binding domain and leucine-rich repeat containing (NLR) family proteins are crucial for immune responses.
• NLRX1, a mitochondrial-associated NLR, modulates immune and metabolic processes.
• Identifying natural ligands for NLRX1 is key to understanding its regulatory functions.

Purpose of the Study:

• To investigate the structure of NLRX1 and identify natural compounds that bind to it.
• To assess the anti-inflammatory potential of identified NLRX1 ligands.
• To elucidate the role of NLRX1 in mediating the anti-inflammatory effects of these compounds.

Main Methods:

• Molecular docking simulations to predict potential NLRX1 ligands.
• Surface plasmon resonance (SPR) spectroscopy to experimentally validate ligand binding.
• Site-directed mutagenesis to identify critical binding residues.
• In vitro assays using bone marrow-derived macrophages (BMDMs) to assess NF-κB pathway activity.
• In vivo studies using a mouse model of dextran sodium sulfate (DSS)-induced colitis.

Main Results:

• Conjugated trienes, polyketides, and various lipids, including punicic acid (PUA), eleostearic acid (ESA), and docosahexaenoic acid (DHA), were predicted to target NLRX1.
• PUA, ESA, and DHA demonstrated binding to the C-terminal fragment of NLRX1 (cNLRX1), with binding affinity validated by SPR.
• Mutagenesis studies identified ASP677, PHE680, PHE681, and GLU684 as critical residues for ligand binding.
• PUA and DHA suppressed NF-κB activity in BMDMs in an NLRX1-dependent manner.
• PUA exhibited NLRX1-dependent anti-inflammatory effects in a DSS-induced colitis mouse model.

Conclusions:

• Novel small molecules, including PUA and DHA, have been identified as NLRX1 ligands.
• These identified ligands exert anti-inflammatory actions through an NLRX1-dependent mechanism involving NF-κB regulation.
• The findings support the therapeutic potential of targeting NLRX1 with natural compounds for inflammatory diseases.