title: Modeling-Enabled Characterization of Novel NLRX1 Ligands.
abstract: Nucleotide-binding domain and leucine-rich repeat containing (NLR) family are intracellular sentinels of cytosolic homeostasis that orchestrate immune and inflammatory responses in infectious and immune-mediated diseases. NLRX1 is a mitochondrial-associated NOD-like receptor involved in the modulation of immune and metabolic responses. This study utilizes molecular docking approaches to investigate the structure of NLRX1 and experimentally assesses binding to naturally occurring compounds from several natural product and lipid databases. Screening of compound libraries predicts targeting of NLRX1 by conjugated trienes, polyketides, prenol lipids, sterol lipids, and coenzyme A-containing fatty acids for activating the NLRX1 pathway. The ligands of NLRX1 were identified by docking punicic acid (PUA), eleostearic acid (ESA), and docosahexaenoic acid (DHA) to the C-terminal fragment of the human NLRX1 (cNLRX1). Their binding and that of positive control RNA to cNLRX1 were experimentally determined by surface plasmon resonance (SPR) spectroscopy. In addition, the ligand binding sites of cNLRX1 were predicted in silico and validated experimentally. Target mutagenesis studies demonstrate that mutation of 4 critical residues ASP677, PHE680, PHE681, and GLU684 to alanine resulted in diminished affinity of PUA, ESA, and DHA to NLRX1. Consistent with the regulatory actions of NLRX1 on the NF-κB pathway, treatment of bone marrow derived macrophages (BMDM)s with PUA and DHA suppressed NF-κB activity in a NLRX1 dependent mechanism. In addition, a series of pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the regulatory function of PUA on colitis is NLRX1 dependent. Thus, we identified novel small molecules that bind to NLRX1 and exert anti-inflammatory actions.
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Pinyi
authors.initials: P
authors.lastname: Lu
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Raquel
authors.initials: R
authors.lastname: Hontecillas
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Vida
authors.initials: V
authors.lastname: Abedi
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Shiv
authors.initials: S
authors.lastname: Kale
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Andrew
authors.initials: A
authors.lastname: Leber
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Chase
authors.initials: C
authors.lastname: Heltzel
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Mark
authors.initials: M
authors.lastname: Langowski
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Victoria
authors.initials: V
authors.lastname: Godfrey
authors.affiliation: BioTherapeutics, 1800 Kraft Drive, Suite 200, Blacksburg, Virginia, 24060, United States of America.
authors.forename: Casandra
authors.initials: C
authors.lastname: Philipson
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Nuria
authors.initials: N
authors.lastname: Tubau-Juni
authors.affiliation: BioTherapeutics, 1800 Kraft Drive, Suite 200, Blacksburg, Virginia, 24060, United States of America.
authors.forename: Adria
authors.initials: A
authors.lastname: Carbo
authors.affiliation: Laboratory of Medicine & Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Stephen
authors.initials: S
authors.lastname: Girardin
authors.affiliation: Georgetown University Medical Center, Washington, District of Columbia, 20057, United States of America.
authors.forename: Aykut
authors.initials: A
authors.lastname: Uren
authors.affiliation: The Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.|Nutritional Immunology and Molecular Medicine Laboratory (www.nimml.org), Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, 24061, United States of America.
authors.forename: Josep
authors.initials: J
authors.lastname: Bassaganya-Riera
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D003092:Colitis / Q000188:drug therapy; D004353:Drug Evaluation, Preclinical; D005227:Fatty Acids / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008024:Ligands; D008042:Linolenic Acids / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D051379:Mice; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D062105:Molecular Docking Simulation; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D000893:Anti-Inflammatory Agents; D005227:Fatty Acids; D008024:Ligands; D008042:Linolenic Acids; D024101:Mitochondrial Proteins; D016328:NF-kappa B; C527006:NLRX1 protein, human; D010446:Peptide Fragments; C519714:punicic acid