7-Hydroxycoumarins Are Affinity-Based Fluorescent Probes for Competitive Binding Studies of Macrophage Migration Inhibitory Factor

Abstract

Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto–enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site ( K i = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF–CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.

Proteolysis Targeting Chimera (PROTAC) for Macrophage Migration Inhibitory Factor (MIF) Has Anti‐Proliferative Activity in Lung Cancer Cells

“…The K i values for the PROTACs of group 2 with a 7‐hydroxy‐3,4‐dihydrobenzoxazin‐2‐one MIF binding core were all around 100 nM. This is very well in line with the potency of their parent inhibitor 3 , which has a K i value of 150 nM [27] . The results demonstrated that our design strategy for linkers did not or minimally perturb target engagement.…”

“…The candidate PROTACs inhibited MIF enzymatic activity with nanomolar inhibition constants ( K i ). The group 1 PROTACs that contain a 7‐hydroxycoumarin MIF binding core provided K i values between 117 to 999 nM, which is in the same range as the K i of MIF inhibitor 2 that was reported to be 370 nM (Table S2) [27] . Interestingly, the PROTACs MD1 – 4 with one carbon atom between the triazole and the amide functionality are more potent MIF tautomerase inhibitors compared to PROTACs with two ( MD5 ) or three carbon atoms ( MD6 ) in this position.…”

“…Previously, our group and others reported structure–activity relationships (SARs) for 7‐hydroxycoumarin derivatives based on inhibitor 2 (Figure 1 A ). [ 27 , 28 ] We also found that compounds containing a 7‐hydroxy‐3,4‐dihydrobenzoxazin‐2‐ones backbone such as 3 can also provide potent inhibition against MIF. [ 27 , 29 ] Furthermore, the Jorgensen lab discovered potent MIF inhibitors that contain a biaryltriazole or pyrazole scaffold.…”

“…[ 27 , 28 ] We also found that compounds containing a 7‐hydroxy‐3,4‐dihydrobenzoxazin‐2‐ones backbone such as 3 can also provide potent inhibition against MIF. [ 27 , 29 ] Furthermore, the Jorgensen lab discovered potent MIF inhibitors that contain a biaryltriazole or pyrazole scaffold. [ 30 , 31 ] However, the potency to inhibit MIF tautomerase activity does not always correlate well with the potency to inhibit the MIF‐CD74 interaction or MIF induced signaling in cell‐based studies.…”

Trending Topics on Coumarin and Its Derivatives in 2020

“…Other reviews report on the use of 7-hydroxycoumarin and its derivatives in determining the activity of cytochromes P450 (CYP) enzymes [ 74 ] as well as 7-aminocoumarin derivatives in determining amino acids from serine or cysteine proteases [ 58 ]. In other cases, they are used to detect the metabolism of mitochondrial cysteines, the oxidation of which is a measure of cellular oxidative stress ( Figure 8 , compound LI ) [ 75 ].…”