Discovery and Anti-Inflammatory Activity of a Cyanobacterial Fatty Acid Targeting the Keap1/Nrf2 Pathway

The monounsaturated fatty acid 7(E)-9-keto-hexadec-7-enoic acid (1) and three structurally related analogues with different oxidation states and degrees of unsaturation (2-4) were discovered from a marine benthic cyanobacterial mat collected from Delta Shoal, Florida Keys. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The structure of 1 contained an alpha,beta-unsaturated carbonyl system, a key motif required for the activation of the Keap1/Nrf2-ARE pathway that is involved in the activation of antioxidant and phase II detoxification enzymes. Compounds 1-4 were screened in ARE-luciferase reporter gene assay using stably transfected HEK293 cells, and only 1 significantly induced Nrf2 activity at 32 and 10 mu M, whereas 2-4 were inactive. As there is crosstalk between inflammation and oxidative stress, subsequent biological studies were focused on 1 to investigate its anti-inflammatory potential. Compound 1 induced Nqo1, a well-known target gene of Nrf2, and suppressed iNos transcript levels, which translated into reduced levels of nitric oxide in LPS-activated mouse macrophage RAW264.7 cells, a more relevant model for inflammation. RNA sequencing was performed to capture the effects of 1 on a global level and identified additional canonical pathways and upstream regulators involved in inflammation and immune response, particularly those related to multiple sclerosis. A targeted survey of marine cyanobacterial samples from other geographic locations, including Guam, suggested the widespread occurrence of 1. Furthermore, the previous isolation of 1 from marine diatoms and green algae implied a potentially important ecological role across marine algal eukaryotes and prokaryotes. The previous isolation from sea lettuce raises the possibility of dietary intervention to attenuate inflammation and related disease progression.