Chemically Modified Peptide Scaffolds Target the CFTR-Associated Ligand PDZ Domain

Abstract

PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR). Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL), and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.

Acetylation of C/EBPα inhibits its granulopoietic function

“…However, the acetylated lysine residues in recently published crystal structures were found to have the H–N–C=O dihedral angles with both 0° and 180° ( Fig. 3b ) 48 49 50 . Thus, the H–N–C=O dihedral angle of all four acetylated lysine residues were set to 180° in the second acetylated model (K2Ac_b-DNA).…”

PDZ Domains as Drug Targets

“…A subset of the 80 peptides was further screened using a positional scan from P 0 to P −10 , with 20 different amino acids obtaining the C-terminal core peptide (P 0 -P −3 ). [298] Substitution of P 0 -P −2 , from Ser-Ile-Ile to Thr-Arg-Val (iCAL36 TRV ), was also shown to yield a modest increase in affinity (Figure 13e,h). The peptide WPTSII resulting from this approach had a modest affinity of 33 µm.…”

Computational Analysis of Energy Landscapes Reveals Dynamic Features that Contribute to Binding of Inhibitors to CFTR-Associated Ligand

“…To facilitate crystallization, kCAL01 was synthesized as a decapeptide (ANSRWQVTRV) containing four N-terminal residues (in italics) that form lattice contacts in other CALP:peptide co-crystals. 1,27,61,62 The kCAL01 decapeptide was synthesized using standard Fmoc solid-phase peptide synthesis and purified by reverse-phase HPLC.…”

“…Previous work 1,26,62 pinpointed "modulator" residues at P -1 , P -3 , and P -4 -P -9 that show individually modest effects on binding and specificity, but together can create significant effects. We compared the CALP:kCAL01 and CALP:iCAL36 protomer A structures in order to determine the effect of these modulator residues on inhibitor binding.…”