Journal: Chembiochem : a European journal of chemical biology

Article Title: N -acyl Amides from Neisseria meningitidis and Their Role in Sphingosine Receptor Signaling

doi: 10.1002/cbic.202200490

Figure Lengend Snippet: Identification of N -acyl amides 1 and 2 from Nm NAT and bioactivity testing. (a) Molecular networking of 20 of the 30 identified N -acyl ornithines (left, navy) and lysines (right, blue), where nodes are connected by tandem-MS patterns. (b) Production levels of 1 and 2 compared to the vector (pET28a) control. (c) New bacterially-derived metabolites 1 ( m/z 359) and 2 ( m/z 373). (d) Dose-dependent activation of S1PR4 by 1 and 2 observed in a PRESTO-Tango assay, quantified as fold over DMSO vehicle control (relative luminescence units (RLU) of compound divided by the DMSO RLU). Positive control ligand sphingosine-1-phosphate (S1P) is shown. (e) Change in percentages of IL-17A+ cells, quantified as fold over DMSO vehicle control in T H 17 polarized cells from an IL-17A Katushka IL-10 eGFP FoxP3 RFP reporter assay system in CD4 + T cells. Error bars represent SD. Two-tailed t-test. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: GPCR screening was performed according to the protocol reported by Chen et al. [ ] Briefly, HTLA cells (a HEK293-derived cell line containing a stably integrated tTa-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin were seeded in 96-well tissue culture plates (Eppendorf) and transfected with 200 ng of S1PR4 plasmid (Addgene plasmid #66499) [ ] for 20 h. The medium was then replaced with Dulbecco’s Modified Eagle Medium (DMEM) with 20 mM N -2-hydroxyethylpiperazine- N -ethanesulfonic acid (HEPES) buffer and 1% Penicillin/Streptomycin, and independently treated with compounds 1 and 2 , sphingosine-1-phosphate (S1P) (Fisher Scientific), and dimethyl sulfoxide (DMSO) solvent vehicle after 2 h. After 20 h of treatment, the luminescence was read by incubating each well for 20 min with 50 μl/well of Bright-Glo solution (Promega) diluted 20-fold in Dulbecco’s phosphate-buffered saline (DPBS) with 20 mM-HEPES.

Techniques: Plasmid Preparation, Control, Derivative Assay, Activation Assay, Positive Control, Reporter Assay, Two Tailed Test