Activity‐Based Probes for 15‐Lipoxygenase‐1

Abstract

Human 15-lipoxygenase-1 (15-LOX-1) plays an important role in several inflammatory lung diseases such as asthma, COPD and chronic bronchitis as well as in various CNS diseases like Alzheimer's, Parkinson's and stroke. Activity-based probes of 15-LOX-1 are required to explore the role of this enzyme further and to enable drug discovery. In this study, we developed the first 15-LOX-1 activity-based probe as an efficient chemical tool for activity-based labeling of recombinant 15-LOX-1 that also provides 15-LOX-1 dependent labeling in cell lysates and tissue samples. Mimicking the natural substrate of the enzyme, we designed activity-based probes that covalently bind to the active enzyme and include a terminal alkene as chemical reporter for bioorthogonal linkage of a detectable functionality via the oxidative Heck reaction. We believe that the activity-based labeling of 15-LOX-1 will enable the investigation and identification of this enzyme in complex biological samples, which opens completely new opportunities for drug discovery. Keywords15-LOX-1; activity-based probes; Kitz-Wilson plots; enzyme kinetics; irreversible inhibition Activity-based protein profiling (ABPP) has become a powerful method for the analysis of enzyme function and the selectivity of enzyme inhibitors in complex disease models.[1] In ABPP, small molecule substrate analogues, known as activity-based probes, are used to covalently bind to the active site of enzymes depending on their activity. Currently, many research groups use ABPP to investigate various enzyme classes, like cysteine proteases [2][3][4], serine hydrolases [5,6] Development of activity-based probes for target enzymes start from irreversible mechanismbased enzyme inhibitors. For soybean lipoxygenase, structural analogs of PUFAs have been reported in which the cis-alkenes are replaced by alkynes that proved to be irreversible inactivators of LOX enzymes. [39,40] Inactivation is expected to proceed through single electron oxidation of the bis-propargylic carbon resulting in an allene radical that is highly reactive and binds covalently to the enzyme's active site. We aimed to use this type of inhibitor as starting structure to develop activity based probes for LOX enzymes.Mimicking the natural 15-LOX-1 substrate, linoleic acid, inhibitors were designed incorporating a bis-alkyne core structure and their binding properties were investigated.[39,40] After modeling studies, in contrast with the previous inhibitors we shift the position of the bis-alkyne moiety from 9,12 to the 5,8 position due to structural differences in the active sites between the two enzymes ( Figure S9) but also aiming to yield less lipophilic compounds. Next, we developed ABPP probes that include both a bis-alkyne functionality for covalent linkage to the active enzyme and a terminal alkene as chemical reporter for bioorthogonal linkage of a detectable functionality (Figure 1). Application of a terminal alkene as chemical reporter and not the more commonly used terminal alkyne enables straightforward ...

Scutellarein alleviates chronic obstructive pulmonary disease through inhibition of ferroptosis by chelating iron and interacting with arachidonate 15‐lipoxygenase

“…Additionally, the iron-chelating activity of scutellarein also decreased the levels of labile Fe 2+ , which may further result in an impaired catalytic activity of ALOX15 (Ivanov et al, 2015;Meng et al, 2018). Significantly, up-regulation of ALOX15 expression was observed in 20%-40% of COPD patients, ALOX15 and its metabolites play an important role in the pathophysiology of COPD (Eleftheriadis et al, 2016;Xu et al, 2021). Therefore, targeting ALOX15 may represent a valuable strategy in the treatment of COPD and airway inflammation.…”

Photoactivation provides a mechanistic explanation for pan-assay interference behaviour of 2-aminopyrroles in lipoxygenase inhibition

“…The screening for h-15-LOX-1 inhibition was done by using a UV absorption assay for the h-15-LOX-1 product, 13(S)-HODE (l max 234 nm) formed by enzymatic conversion from linoleic acid [27,32]. This assay was performed in a 96-well format, which is well suited for medium-throughput screening, IC 50 measurements and enzyme kinetics studies.…”

Activity‐Based Protein Profiling of Oxidases and Reductases

“…The group of Dekker has designed the ABPP probe N144 ( 22 ), which mimics the natural polyunsaturated fatty acid substrate but contains a bispropargylic warhead (Scheme 5). [63] Single‐electron oxidation of the bis‐propargylic carbon atom results in a highly reactive allene radical, which leads to the covalent attachment of the probe. For this original design Dekker et al.…”