Abstract
Protein–protein interactions have become attractive targets for both experimental and therapeutic interventions. The PSD-95/Dlg1/ZO-1 (PDZ) domain is found in a large family of eukaryotic scaffold proteins that plays important roles in intracellular trafficking and localization of many target proteins. Here, we seek inhibitors of the PDZ protein that facilitates post-endocytic degradation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR): the CFTR-associated ligand (CAL). We develop and validate biochemical screens and identify methyl-3,4-dephostatin (MD) and its analog ethyl-3,4-dephostatin (ED) as CAL PDZ inhibitors. Depending on conditions, MD can bind either covalently or non-covalently. Crystallographic and NMR data confirm that MD attacks a pocket at a site distinct from the canonical peptide-binding groove, and suggests an allosteric connection between target residue Cys319 and the conserved Leu291 in the GLGI motif. MD and ED thus appear to represent the first examples of small-molecule allosteric regulation of PDZ:peptide affinity. Their mechanism of action may exploit the known conformational plasticity of the PDZ domains and suggests that allosteric modulation may represent a strategy for targeting of this family of protein–protein binding modules.
Highlights
Protein–protein interactions play crucial roles in many biological processes, both normal and pathological
We reported the stereochemical validation of one inhibitor scaffold and its interaction with CF transmembrane conductance regulator (CFTR)-associated ligand (CAL), which suggested a mechanism of small-molecule inhibition not previously seen for PDZ domains
To identify small molecules that could disrupt the interaction between PDZ-binding motifs and CAL, we designed high-throughput compatible assays using our peptide inhibitor iCAL36 (ANSRWPTSII) as a reporter for CALP binding [19,33]
Summary
Protein–protein interactions play crucial roles in many biological processes, both normal and pathological. They are attractive targets for both experimental and therapeutic interventions. Such interactions can be difficult to disrupt, due to their distributed contact surfaces and shallow pockets. In the past two decades, much effort has been devoted to understand such interactions and targeting them therapeutically. Success has been achieved by targeting protein binding sites directly. Complex chemistries such as linear peptides, macrocycles, or more complex secondary and tertiary structures can mediate the relatively extensive interactions that are needed to disrupt the binding interface [1,2]
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