Design of Tracers in Fluorescence Polarization Assay for Extensive Application in Small Molecule Drug Discovery.

Development of fluorescence polarization (FP) assays, especially in a competitive manner, is a potent and mature tool for measuring the binding affinities of small molecules. This approach is suitable for high-throughput screening (HTS) for initial ligands and is also applicable for further study of the structure-activity relationships (SARs) of candidate compounds for drug discovery. Buffer and tracer, especially rational design of the tracer, play a vital role in an FP assay system. In this perspective, we provided different kinds of approaches for tracer design based on successful cases in recent years. We classified these tracers by different types of ligands in tracers, including peptide, nucleic acid, natural product, and small molecule. To make this technology accessible for more targets, we briefly described the basic theory and workflow, followed by highlighting the design and application of typical FP tracers from a perspective of medicinal chemistry.

Protein Phosphatase 5-Recruiting Chimeras for Accelerating Apoptosis-Signal-Regulated Kinase 1 Dephosphorylation with Antiproliferative Activity.

A normal phosphorylation state is essential for the function of proteins. Biased regulation frequently results in morbidity, especially for the hyperphosphorylation of oncoproteins. The hyperphosphorylation of ASK1 at Thr838 leads to a persistently high activity state, which accelerates the course of gastric cancer. Under normal conditions, PP5 specifically dephosphorylates p-ASK1T838 in cells, thereby weakening ASK1 to a low-basal activity state. However, in tumor types, PP5 shows low activity with a self-inhibition mechanism, making p-ASK1T838 remain at a high level. Thus, we aim to design phosphatase recruitment chimeras (PHORCs) through a proximity-mediated effect for specifically accelerating the dephosphorylation of p-ASK1T838. Herein, we describe DDO3711 as the first PP5-recruiting PHORC, which is formed by connecting a small molecular ASK1 inhibitor to a PP5 activator through a chemical linker, to effectively decrease the level of p-ASK1T838 in vitro and in vivo. DDO3711 shows preferable antiproliferative activity (IC50 = 0.5 µM) against MKN45 cells through a direct binding and proximity-mediated mechanism, while the ASK1 inhibitor and the PP5 activator, used alone or in combination, exhibit no effect on MKN45 cells. Using DDO3711, PHORCs are identified as effective tools to accelerate the dephosphorylation of POIs and provide important evidence to achieve precise phosphorylation regulation, which will promote confidence in the further regulation of abnormally phosphorylated oncoproteins.

Beyond Proteolysis-Targeting Chimeric Molecules: Designing Heterobifunctional Molecules Based on Functional Effectors.

In recent years, with the successful development of proteolysis-targeting chimeric molecules (PROTACs), the potential of heterobifunctional molecules to contribute to reenvisioning drug design, especially small-molecule drugs, has been increasingly recognized. Inspired by PROTACs, diverse heterobifunctional molecules have been reported to simultaneously bind two or more molecules and bring them into proximity to interaction, such as ribonuclease-recruiting, autophagy-recruiting, lysosome-recruiting, kinase-recruiting, phosphatase-recruiting, glycosyltransferase-recruiting, and acetyltransferase-recruiting chimeras. On the basis of the heterobifunctional principle, more opportunities for advancing drug design by linking potential effectors to a protein of interest (POI) have emerged. Herein, we introduce heterobifunctional molecules other than PROTACs, summarize the limitations of existing molecules, list the main challenges, and propose perspectives for future research directions, providing insight into alternative design strategies based on substrate-proximity-based targeting.