Discovery and Development of Cyclic Peptide Inhibitors of CIB1

Abstract

Calcium and integrin binding protein 1 (CIB1) is a small, intracellular protein recently implicated in survival and proliferation of triple-negative breast cancer (TNBC). Considering its interactions with PAK1 and downstream signaling, CIB1 has been suggested as a potential therapeutic target in TNBC. As such, CIB1 has been the focus of inhibitor discovery efforts. To overcome issues of potency and stability in previously reported CIB1 inhibitors, we deploy mRNA display to discover new cyclic peptide inhibitors with improved biophysical properties and cellular activity. We advance UNC10245131, a cyclic peptide with low nanomolar affinity and good selectivity for CIB1 over other EF-hand domain proteins and improved permeability and stability over previously identified linear peptide inhibitor UNC10245092. Unlike UNC10245092, UNC10245131 lacks cytotoxicity and does not affect downstream signaling. Despite this, UNC10245131 is a potent ligand that could aid in clarifying roles of CIB1 in TNBC survival and proliferation and other CIB1-associated biological phenotypes.

Figure 1

mRNA display results. (A) Outline of mRNA display selection process. (B) S_N2 reaction between C-terminal cysteine residue and N-terminal ClAcY residue to form thioether cyclization linkage during translation. (C) Families of CIB1 binding peptides are highlighted in blue. Bold peptides are representative hits from each family analyzed in this study. An asterisk indicates a detected stop codon. Red indicates an amino acid mutation from the bold peptide. (D) IC50 values for the bold peptides from panel C as determined by TR-FRET against CIB1.

Figure 2

Structure and activity of UNC10245131. (A) Table of IC50 values for alanine mutants of UNC10245131; data represent average and standard deviation of a minimum of six replicates. (B) Interaction map of UNC10245131 when docked into the CIB1 binding pocket. (C) Structure of UNC10245092 (phage peptide) bound to CIB1 (PDB 6OD0) and (D) UNC10245092 bound to CIB1; dotted lines represent the interactions between Y10 and W2 with CIB1 residues K107, F115, and S160. (E) UNC10245131 docked into the CIB1 binding pocket and (F) UNC10245131 docked into CIB1; dotted lines represent the interactions of Y1 and W10 with CIB1 residues K107, S160, and F173.

Figure 3

Cellular activity, target engagement, and proteomic pulldown. (A) Western blots of 6 h- (left) and 24 h-treated (right) MDA-MB-231 and MDA-MB-468 cells. (B) UNC10245092 and UNC10245131, immobilized via streptavidin–biotin interaction, pulled down CIB1 from MDA436 cell lysates. (C) Venn diagrams indicating the number of proteins found in 1, 2, 3, or 4 replicates of the proteomic pulldown. Colors of the Venn diagram each indicate a unique replicate.