Abstract
AbstractLipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity‐based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one‐step activity‐based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA‐based assay for affinity capture and antibody‐based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity‐based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain‐like consensus motif in 15‐LOX‐1. This indicates that 15‐LOX‐1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications.
Highlights
Small molecule modulation of enzyme activity is an important strategy in drug discovery
The biotinylated bisalkynes were subjected to 15-LOX-1 binding studies using a 15-LOX-1 activity assay based on the UV-absorbance change for linoleic acid conversion (Figure 1 a)
We found that PD146176 efficiently attenuated the Labelox B mediated LOXs labeling with an inhibiting concentration 50 % (IC50) around 15 mM and a Hill slope around 1
Summary
Small molecule modulation of enzyme activity is an important strategy in drug discovery. Measurement of the activity of specific isoenzymes from the relevant endogenous sources remains challenging. This is true for Lipoxygenases (LOXs), which is a group of enzymes known to be involved in oxidative metabolism of polyunsaturated fatty acids (PUFAs),[1] such as arachidonic acid (AA), and linoleic acid (LA). LOXs are classified according to the position of O2 insertion in AA as 5-LOX, 12LOX or 15-LOX.[2] LOX activity provides a range of products, such as hydroperoxyl eicosatetraenoic acids (HPETEs), hydroxy eicosatetraenoic acids (HETEs), and [+] These authors contributed to this work.
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