Identification of Novel Fragments Binding to the PDZ1‐2 Domain of PSD‐95

Abstract

Inhibition of PSD‐95 has emerged as a promising strategy for the treatment of ischemic stroke, as shown with peptide‐based compounds that target the PDZ domains of PSD‐95. In contrast, developing potent and drug‐like small molecules against the PSD‐95 PDZ domains has so far been unsuccessful. Here, we explore the druggability of the PSD‐95 PDZ1‐2 domain and use fragment screening to investigate if this protein is prone to binding small molecules. We screened 2500 fragments by fluorescence polarization (FP) and validated the hits by surface plasmon resonance (SPR), including an inhibition counter‐test, and found four promising fragments. Three ligand efficient fragments were shown by 1H,15N HSQC NMR to bind in the small hydrophobic P0 pockets of PDZ1‐2, and one of them underwent structure‐activity relationship (SAR) studies. Overall, we demonstrate that fragment screening can successfully be applied to PDZ1‐2 of PSD‐95 and disclose novel fragments that can serve as starting points for optimization towards small‐molecule PDZ domain inhibitors.

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

“…Interestingly, it has been a trend for researchers to choose FP as the only or involved screening assay combined with other classical screening assays to identify hits from a large fragment library in recent years. For example, Zang et al employed FP during the first screening of 2500 fragments for small molecules binding to the PDZ1-2 domain of PSD-95 in 2021 . Sixty-five primary hits with 2.5% hit rate reversed the previous thought that PDZ domains are nondruggable.…”

“…For example, Zang et al employed FP during the first screening of 2500 fragments for small molecules binding to the PDZ1-2 domain of PSD-95 in 2021. 129 Sixty-five primary hits with 2.5% hit rate reversed the previous thought that PDZ domains are nondruggable. This result showed that FP could be developed into a robust, potential, and promising fragment screening method.…”