Forrestiacids A (1) and B (2) are a novel class of [4+2] type pentaterpenoids derived from a rearranged lanostane moiety (dienophile) and an abietane unit (diene). These unprecedented molecules were isolated using guidance by molecular ion networking (MoIN) from Pseudotsuga forrestii, an endangered member of the Asian Douglas Fir Family. The intermolecular hetero-Diels-Alder adducts feature an unusual bicyclo[2.2.2]octene ring system. Their structures were elucidated by spectroscopic analysis, GIAO NMR calculations and DP4+ probability analyses, electronic circular dichroism calculations, and X-ray diffraction analysis. This unique addition to the pentaterpene family represents the largest and the most complex molecule successfully assigned using computational approaches to predict accurately chemical shift values. Compounds 1 and 2 exhibited potent inhibitory activities (IC50 s <5 μM) of ATP-citrate lyase (ACL), a new drug target for the treatment of glycolipid metabolic disorders including hyperlipidemia. Validating this activity 1 effectively attenuated the de novo lipogenesis in HepG2 cells. These findings provide a new chemical class for developing potential therapeutic agents for ACL-related diseases with strong links to traditional medicines.
Malic enzyme (ME) plays a vital role in determining the extent of lipid accumulation in oleaginous fungi being the major provider of NADPH for the activity of fatty acid synthase (FAS). We report here the first direct evidence of the existence of a lipogenic multienzyme complex (the lipid metabolon) involving ME, FAS, ATP: citrate lyase (ACL), acetyl-CoA carboxylase (ACC), pyruvate carboxylase (PC) and malate dehydrogenase (MDH) in Cunninghamella bainieri 2A1. Cell-free extracts prepared from cells taken in both growth and lipid accumulation phases were prepared by protoplasting and subjected to Blue Native (BN)-PAGE coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A high molecular mass complex (approx. 3.2 MDa) consisting of the above enzymes was detected during lipid accumulation phase indicating positive evidence of multienzyme complex formation. The complex was not detected in cells during the balanced phase of growth or when lipid accumulation ceased, suggesting that it was transiently formed only during lipogenesis.