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

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 (Ki = 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.

Optimized Inhibitors of MDM2 via an Attempted Protein-Templated Reductive Amination.

Innovative and efficient hit-identification techniques are required to accelerate drug discovery. Protein-templated fragment ligations represent a promising strategy in early drug discovery, enabling the target to assemble and select its binders from a pool of building blocks. Development of new protein-templated reactions to access a larger structural diversity and expansion of the variety of targets to demonstrate the scope of the technique are of prime interest for medicinal chemists. Herein, we present our attempts to use a protein-templated reductive amination to target protein-protein interactions (PPIs), a challenging class of drug targets. We address a flexible pocket, which is difficult to achieve by structure-based drug design. After careful analysis we did not find one of the possible products in the kinetic target-guided synthesis (KTGS) approach, however subsequent synthesis and biochemical evaluation of each library member demonstrated that all the obtained molecules inhibit MDM2. The most potent library member (Ki =0.095 µm) identified is almost as active as Nutlin-3, a potent inhibitor of the p53-MDM2 PPI.

Replacement of an Indole Scaffold Targeting Human 15-Lipoxygenase-1 Using Combinatorial Chemistry.

Human 15-lipoxygenase-1 (15-LOX-1) belongs to the class of lipoxygenases, which catalyze oxygenation of polyunsaturated fatty acids, such as arachidonic and linoleic acid. Recent studies have shown that 15-LOX-1 plays an important role in physiological processes linked to several diseases such as airway inflammation disease, coronary artery disease, and several types of cancer such as rectal, colon, breast and prostate cancer. In this study, we aimed to extend the structural diversity of 15-LOX-1 inhibitors, starting from the recently identified indolyl core. In order to find new scaffolds, we employed a combinatorial approach using various aromatic aldehydes and an aliphatic hydrazide tail. This scaffold-hopping study resulted in the identification of the 3-pyridylring as a suitable replacement of the indolyl core with an inhibitory activity in the micromolar range (IC 50=16±6 µm) and a rapid and efficient structure-activity relationship investigation.