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.
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This work was supported by the National Institutes of Health [grant numbers R01-DK101541, P20-GM113132, R21-NS067613, T32-GM008704, P30-GM106394, P30-DK117469]; the Cystic Fibrosis Foundation [grant number STANTO15R0]; the Neukom CompX award; the American Lebanese Syrian Associated Charities (ALSAC); the St. Jude Children’s Research Hospital; beamline access was supported in part by the NIH [grant numbers GM-0080, P41-GM111244, P41-GM103393]; and the DOE [grant numbers DE-SC0012704, DE-AC02-76SF00515].
Zhao, Yu; Cushing, Patrick R.; Smithson, David C.; Pellegrini, Maria; Pletnev, Alexandre A.; Al-Ayyoubi, Sahar; Grassetti, Andrew V.; Gerber, Scott A.; Guy, R. Kiplin; and Madden, Dean R., "Cysteine Modifiers Suggest an Allosteric Inhibitory Site on the CAL PDZ Domain" (2018). Pharmaceutical Sciences Faculty Publications. 113.