Date of Award

2023

Document Type

Thesis (Ph.D.)

Department or Program

Biochemistry and Cell Biology

First Advisor

Dean R. Madden

Abstract

Significant therapeutic achievements have been made in the past decade towards increasing the pool of correctly folded F508del-CFTR and enhancing its activity at the apical membrane. Despite this success, there are still no FDA-approved therapeutics that directly target the post-endocytic stability of F508del-CFTR. Upon internalization, it is rapidly degraded through binding the CFTR-associated ligand (CAL) PDZ domain (CALP), which triggers its degradation in the lysosome. Our group and others have shown that inhibiting CAL increases the half-life of functional F508del-CFTR at the cell‑membrane and significantly enhances ion transport, suggesting that ‘stabilizer’ compounds, especially in conjunction with current CF therapeutics, would augment treatment efficacy.

Unfortunately, previous efforts to design inhibitors of CALP yielded high specificity peptides with weak affinity (iCAL36L; KI = ~40-60 µM) and a low potency small-molecule inhibitor that covalently modifies Cys319 of CALP. To enhance target affinity without sacrificing specificity, we designed peptidomimetic inhibitors containing modified lysine scaffolds that were able to bind distal regions outside of the conserved peptide-binding cleft. We show by structure-activity relationships that targeting the β1-β2 loop confers enhanced affinity for CALP while likely conserving specificity. We also identified a potent Cys319 modifier that modulates both the thermostability and peptide‑binding affinity of CALP through a multi-step reaction involving multiple adducted states. The inhibitory effect is mediated through a non-covalent, allosteric interaction between two adducted CALP states. These data provide insight into CALP protein dynamics and offer a new strategy for designing CALP inhibitors.

Download

Share

COinS