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PDZ (PSD-95, Dlg, ZO-1) domains are ubiquitous interaction modules that are involved in many cellular signal transduction pathways. Interference with PDZ-mediated protein-protein interactions has important implications in disease-related signaling processes. For this reason, PDZ domains have gained attention as potential targets for inhibitor design and, in the long run, drug development. Herein we report the development of small molecules to probe the function of the PDZ domain from human AF6 (ALL1-fused gene from chromosome 6), which is an essential component of cell-cell junctions. These compounds bind to AF6 PDZ with substantially higher affinity than the peptide (Ile-Gln-Ser-Val-Glu-Val) derived from its natural ligand, EphB2. In intact cells, the compounds inhibit the AF6-Bcr interaction and interfere with epidermal growth factor (EGF)-dependent signaling., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Shank is the central scaffolding protein of the postsynaptic density (PSD) protein complex found in cells of the central nervous system. Cellular studies indicate a prominent role of the protein in the organization of the PSD, in the development of neuronal morphology, in neuronal signaling, and in synaptic plasticity, thus linking Shank functions to the molecular basis of learning and memory. Mutations in the Shank gene have been found in several neuronal disorders including mental retardation, typical autism, and Asperger syndrome. Shank is linked to the PSD complex via its PDZ domain that binds to the C-terminus of guanylate-kinase-associated protein (GKAP). Here, small-molecule inhibitors of Shank3 PDZ domain are developed. A fluorescence polarization assay based on an identified high-affinity peptide is established, and tetrahydroquinoline carboxylates are identified as inhibitors of this protein-protein interaction. Chemical synthesis via a hetero-Diels-Alder strategy is employed for hit optimization, and structure-activity relationship studies are performed. Best hits possess K(i) values in the 10 μM range, and binding to the PDZ domain is confirmed by ¹H,¹⁵N HSQC NMR experiments. One of the hits crystallizes with the Shank3 PDZ domain. The structure, analyzed at a resolution of 1.85 Å, reveals details of the binding mode. Finally, binding to PDZ domains of PSD-95, syntrophin, and DVL3 was studied using ¹H,¹⁵N HSQC NMR spectroscopy., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)