Structure of New Ferroverdins Recruiting Unconventional Ferrous Iron Chelating Agents

Ferroverdins are ferrous iron (Fe²⁺)-nitrosophenolato complexes produced by a few Streptomyces species as a response to iron overload. Previously, three ferroverdins were identified: ferroverdin A, in which three molecules of p-vinylphenyl-3-nitroso-4-hydroxybenzoate (p-vinylphenyl-3,4-NHBA) are recruited to bind Fe²⁺, and Ferroverdin B and Ferroverdin C, in which one molecule of p-vinylphenyl-3,4-NHBA is substituted by hydroxy-p-vinylphenyl-3,4-NHBA, and by carboxy-p-vinylphenyl-3,4-NHBA, respectively. These molecules, especially ferroverdin B, are potent inhibitors of the human cholesteryl ester transfer protein (CETP) and therefore candidate hits for the development of drugs that increase the serum concentration of high-density lipoprotein cholesterol, thereby diminishing the risk of atherosclerotic cardiovascular disease. In this work, we used high-resolution mass spectrometry combined with tandem mass spectrometry to identify 43 novel ferroverdins from the cytosol of two Streptomyces lunaelactis species. For 13 of them (designated ferroverdins C2, C3, D, D2, D3, E, F, G, H, CD, DE, DF, and DG), we could elucidate their structure, and for the other 17 new ferroverdins, ambiguity remains for one of the three ligands. p-formylphenyl-3,4-NHBA, p-benzoic acid-3,4-NHBA, 3,4-NHBA, p-phenylpropionate-3,4-NHBA, and p-phenyacetate-3,4-NHBA were identified as new alternative chelators for Fe²⁺-binding, and two compounds (C3 and D3) are the first reported ferroverdins that do not recruit p-vinylphenyl-3,4-NHBA. Our work thus uncovered putative novel CETP inhibitors or ferroverdins with novel bioactivities.