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.

NLRX1 dampens oxidative stress and apoptosis in tissue injury via control of mitochondrial activity

“…A recent study identified the polyunsaturated fatty acid docosahexaenoic acid (DHA) as one of three fatty acid ligands for NLRX1 ( Lu et al, 2015 ). Therefore we examined if DHA could reduce apoptosis after reoxygenation in an NLRX1-dependent fashion.…”

“…Surface plasmon resonance spectroscopy revealed that the polyunsaturated fatty acids DHA, punicic acid, and eleostearic acid bind the C-terminal end of the leucine-rich repeat domain of NLRX1 and modulate intestinal injury in an NLRX1-dependent way ( Lu et al, 2015 ). These findings indicate that NLRX1 activity can be modulated by fatty acid levels.…”

“…The lack of pharmacological activators or inhibitors is therefore a limitation of the present study. Concerning the other NLRX1 ligands described by Lu et al (2015) , we were unable to find a reliable commercial source for punicic acid without contaminating impurities, and we did not examine eleostearic acid, because the original study did not fully investigate how this fatty acid acts on intestinal injury compared with DHA and punicic acid.…”

NLR-Dependent Regulation of Inflammation in Multiple Sclerosis

“…Ligands for NLRX1 and NLRP12 have remained poorly described. However, a number of NLRX1 binding molecules and inhibitors were recently identified using a molecular docking approach to screen natural products and lipid databases ( 193 ). This study by Lu et al revealed that punicic acid (PUA), eleostearic acid (ESA), and docosahexaenoic acid (DHA) can bind to the C-terminal fragment of the human NLRX1.…”

mutLBSgeneDB: mutated ligand binding site gene DataBase

“…Lastly we searched the optimal fit of each drug into targets. The details about this method were described in previous studies (34,35). …”